Bis(thio-hydrazide amide) salts for treatment of cancers

ABSTRACT

Disclosed are bis(thio-hydrazide amide) disalts, which are represented by Structural Formula (I): 
     
       
         
         
             
             
         
       
     
     Y is a covalent bond or a substituted or unsubstituted straight chained hydrocarbyl group. R 1 -R 4  are independently —H, an aliphatic group, a substituted aliphatic group, an aryl group or a substituted aryl group, or R 1  and R 3  taken together with the carbon and nitrogen atoms to which they are bonded, and/or R 2  and R 4  taken together with the carbon and nitrogen atoms to which they are bonded, form a non-aromatic heterocyclic ring optionally fused to an aromatic ring. Z is —O or —S. M +  is a pharmaceutically acceptable monovalent cation and M 2+  is a pharmaceutically acceptable divalent cation. 
     Also, disclosed are pharmaceutical compositions comprising a bis(thio-hydrazide amide) disalt described above. Further disclosed are methods of treating a subject with cancer. The methods comprise the step of administering an effective amount of a bis(thio-hydrazide amide) disalt described above.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 60/582,596, filed Jun. 23, 2004 and U.S.Provisional Patent Application Ser. No. 60/681,368, filed May 16, 2005.The entire teachings of the above applications are incorporated hereinby reference.

BACKGROUND OF THE INVENTION

Many drugs are now available to be used in the treatment of cancer.However, in many cases the cancer fails to respond to the anti-cancertherapy or its growth and/or metastasis is only slowed. Even when atumor initially responds to an anti-cancer therapy by decreasing in sizeor going into remission, the tumor often develops resistance to thedrug. For these reasons, there has been a need for new anti-canceragents and for new drugs which can be used to treat multi-drugresistance cancers.

Certain bis(thio-hydrazide amide) compounds have been described by thepresent inventors as being significantly cytotoxic to cancer cells,including cancer cells that have become multi-drug resistant, and forenhancing the anti-cancer activity of other anti-cancer agents, such aspaclitaxel and Epothilone D (see, e.g., U.S. Publication Nos.2004/0225016 A1, 2003/0045518 and 2003/0119914, the entire contents ofwhich are incorporated herein by reference).

SUMMARY OF THE INVENTION

It has now been found that bis(thio-hydrazide amide) disalts showunexpectedly high water solubility and bioavailability. For example,disodium and dipotassium salts of Compounds (1) and (2), show watersolubility of greater than 1,000 mg/ml, compared with a solubility ofabout 0.1 mg/ml for the corresponding neutral form of Compounds (1) and(2) (see Examples 2, 10 and 15). Similar increases in solubility wereobserved for the disodium and dipotassium salts of Compounds (12), (13)and (14) (see Examples 12-15). Moreover, the bioavailability of thedisodium salt of Compound (1) was 80%, whereas the bioavailability ofthe neutral compound was 4.8% (see Example 16). A representativetautomeric structure of Compounds (1) and (2) are shown below:

Based on these results, novel bis(thio-hydrazide amide) disalts,pharmaceutical compositions comprising a bis(thio-hydrazide amide)disalt and methods of treatment using a bis(thio-hydrazide amide) disaltare disclosed.

One embodiment of the present invention is a compound represented by thefollowing Structural Formula (I), and its tautomeric forms:

Y is a covalent bond or a substituted or unsubstituted straight chainedhydrocarbyl group. R₁-R₄ are independently —H, an aliphatic group, asubstituted aliphatic group, an aryl group or a substituted aryl group,or R₁ and R₃ taken together with the carbon and nitrogen atoms to whichthey are bonded, and/or R₂ and R₄ taken together with the carbon andnitrogen atoms to which they are bonded, form a non-aromaticheterocyclic ring optionally fused to an aromatic ring. Z is —O or —S.M⁺ is a pharmaceutically acceptable monovalent cation and M²⁺ is apharmaceutically acceptable divalent cation.

Another embodiment of the present invention is a pharmaceuticalcomposition comprising a bis(thio-hydrazide amide) disalt disclosedherein and a pharmaceutically acceptable carrier or diluent. Thepharmaceutical compositions can be used in therapy, for example, asanti-cancer agents.

The present invention also provides for a method of treating a subjectwith a cancer. The method comprises administering to the subject aneffective amount of a bis(thio-hydrazide amide) disalt disclosed herein.The bis(thio-hydrazide amide) disalt is administered as a mono-therapy(i.e., as the only anti-cancer drug administered to the subject) or isco-administered with one or more other anti-cancer drugs.

The use of the bis(thio-hydrazide amide) disalts disclosed herein in themanufacture of a medicament for the purpose of treating cancer in anindividual is also provided in the present invention.

The present invention also provides for a method of preparing abis(thio-hydrazide amide) disalt. The method includes the steps ofcombining a neutral bis(thio-hydrazide amide), an organic solvent and abase to form a bis(thio-hydrazide amide) solution; and combining thesolution and an organic antisolvent, thereby precipitating a disalt ofthe bis(thio-hydrazide amide).

In various embodiments, a method of preparing a bis(thio-hydrazideamide) disalt includes the steps of combining a neutralbis(thio-hydrazide amide) and an organic solvent selected from methanol,ethanol, acetone, and methyl ethyl ketone to make a mixture; adding atleast two equivalents of a base selected from sodium hydroxide,potassium hydroxide, sodium methoxide, potassium methoxide, sodiumethoxide and potassium ethoxide to the mixture, thereby forming asolution; and combining the solution and an organic antisolvent selectedfrom pentane, hexane, cyclohexane, heptane, petroleum ether, ethylacetate, and diethyl ether to precipitate the disalt of thebis(thio-hydrazide amide).

In various embodiments, a method of preparing a bis(thio-hydrazideamide) disalt, includes the steps of combining a neutralbis(thio-hydrazide amide), an organic solvent and a base to form abis(thio-hydrazide amide) solution; and separating a disalt of thebis(thio-hydrazide amide).

In various embodiments, a method of preparing a bis(thio-hydrazideamide) disalt includes the steps of combining a neutralbis(thio-hydrazide amide), an organic solvent and a base to form abis(thio-hydrazide amide) solution; and combining the solution andmethyl tert-butyl ether, thereby precipitating a disalt of thebis(thio-hydrazide amide).

In various embodiments, a method of preparing a bis(thio-hydrazideamide) disalt includes the steps of combining a neutralbis(thio-hydrazide amide) and an organic solvent selected from methanol,ethanol, acetone, and methyl ethyl ketone to make a mixture; adding atleast two equivalents of a base selected from sodium hydroxide,potassium hydroxide, sodium methoxide, potassium methoxide, sodiumethoxide and potassium ethoxide to the mixture, thereby forming asolution; and combining the solution and methyl tert-butyl ether toprecipitate the disalt of the bis(thio-hydrazide amide).

Because the disclosed bis(thio-hydrazide amide) disalts have excellentwater solubility and high bioavailability, they can be used inwater-based formulations suitable for intravenous and oraladministration. In addition, the disclosed bis(thio-hydrazide amide)disalts are relatively non-toxic, which allows the use of the discloseddisalts at relatively high doses with minimal side effects. The highwater solubility of the compounds, in turn, makes high dose formulationspossible.

The bis(thio-hydrazide amide) disalts disclosed herein can be used totreat cancers, including cancers that have become multi-drug resistant.Thus, the disclosed bis(thio-hydrazide amide) disalts can be used totreat cancers where other drug regimens have either failed or becomeineffective. Additionally, the disclosed bis(thio-hydrazide amide)disalts are particularly effective when used in combination with otheranti-cancer drugs such as paclitaxel or an analog of paclitaxel.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the structure of paclitaxel (TAXOL).

FIG. 2 is the structure of docetaxel(TAXOTERE).

FIGS. 3-23 are each the structure of a paclitaxel analog.

FIG. 24 is the structure of a polymer comprising a paclitaxel analoggroup pendent from the polymer backbone. The polymer is a terpolymer ofthe three monomer units shown.

DETAILED DESCRIPTION OF THE INVENTION

The bis(thio-hydrazide amide) disalts of the present invention arerepresented by Structural Formula (I).

M⁺ is a pharmaceutically acceptable monovalent cation. M²⁺ is apharmaceutically acceptable divalent cation. “Pharmaceuticallyacceptable” means that the cation is suitable for administration to asubject. Examples of M⁺ or M²⁺ include Li⁺, Na⁺, K⁺, Mg²⁺, Ca²⁺, Zn²⁺,and NR₄ ⁺, wherein each R is independently hydrogen, a substituted orunsubstituted aliphatic group (e.g., a hydroxyalkyl group, aminoalkylgroup or ammoniumalkyl group) or substituted or unsubstituted arylgroup, or two R groups, taken together, form a substituted orunsubstituted non-aromatic heterocyclic ring optionally fused to anaromatic ring. Preferably, the pharmaceutically acceptable cation isLi⁺, Na⁺, K⁺, NH₃(C₂H₅OH)⁺, N(CH₃)₃(C₂H₅OH)⁺, arginine or lysine. Morepreferably, the pharmaceutically acceptable cation is Na⁺ or K⁺. Na⁺ iseven more preferred.

In Structural Formula (I), Z is preferably —O. More preferably, Z is —O;R₁ and R₂ are the same; and R₃ and R₄ are the same.

In one embodiment, Y in Structural Formula (I) is a covalent bond,—C(R₅R₆)—, —(CH₂CH₂)—, trans-(CH═CH)—, cis-(CH═CH)— or —(CC)— group,preferably —C(R₅R₆)—. R₁-R₄ are as described above for StructuralFormula (I). R₅ and R₆ are each independently —H, an aliphatic orsubstituted aliphatic group, or R₅ is —H and R₆ is a substituted orunsubstituted aryl group, or, R₅ and R₆, taken together, are a C2-C6substituted or unsubstituted alkylene group. The pharmaceuticallyacceptable cation is as described above.

In a preferred embodiment of the present invention, thebis(thio-hydrazide amide) disalt is represented by Structural Formula(II):

R₁-R₆ and the pharmaceutically acceptable cation are as described abovefor Structural Formula (I).

In one more preferred embodiment of the present invention, thebis(thio-hydrazide amide) disalt is represented by Structural Formula(II) where R₁ and R₂ are each a substituted or unsubstituted aryl group,preferably a substituted or unsubstituted phenyl group; R₃ and R₄ areeach a substituted or unsubstituted aliphatic group, preferably an alkylgroup, more preferably, methyl or ethyl; and R₅ and R₆ are as describedabove, but R₅ is preferably —H and R₆ is preferably —H, an aliphatic orsubstituted aliphatic group.

Alternatively, R₁ and R₂ are each a substituted or unsubstituted arylgroup; R₃ and R₄ are each a substituted or unsubstituted aliphaticgroup; R₅ is —H; and R₆ is —H, an aliphatic or substituted aliphaticgroup. Preferably, R₁ and R₂ are each a substituted or unsubstitutedaryl group; R₃ and R₄ are each an alkyl group; and R₅ is —H and R₆ is —Hor methyl. Even more preferably, R₁ and R₂ are each a substituted orunsubstituted phenyl group; R₃ and R₄ are each methyl or ethyl; and R₅is —H and R₆ is —H or methyl. Suitable substituents for an aryl grouprepresented by R₁ and R₂ and an aliphatic group represented by R₃, R₄and R₆ are as described below for aryl and aliphatic groups.

In a second more preferred embodiment of the present invention, thebis(thio-hydrazide amide) disalt is represented by Structural Formula(II) where R₁ and R₂ are each a substituted or unsubstituted aliphaticgroup, preferably a C3-C8 cycloalkyl group optionally substituted withat least one alkyl group, more preferably cyclopropyl or1-methylcyclopropyl; R₃ and R₄ are as described above for StructuralFormula (I), preferably both a substituted or unsubstituted alkyl group;and R₅ and R₆ are as described above, but R₅ is preferably —H and R₆ ispreferably —H, an aliphatic or substituted aliphatic group, morepreferably —H or methyl.

Alternatively, the bis(thio-hydrazide amide) disalt is represented byStructural Formula (II) where R₁ and R₂ are each a substituted orunsubstituted aliphatic group; R₃ and R₄ are as described above forStructural Formula (I), preferably both a substituted or unsubstitutedalkyl group; and R₅ is —H and R₆ is —H or an optionally substitutedaliphatic group. Preferably, R₁ and R₂ are both a C3-C8 cycloalkyl groupoptionally substituted with at least one alkyl group; R₃ and R₄ are bothas described above for Structural Formula (I), preferably an alkylgroup; and R₅ is —H and R₆ is —H or an aliphatic or substitutedaliphatic group. More preferably, R₁ and R₂ are both a C3-C8 cycloalkylgroup optionally substituted with at least one alkyl group; R₃ and R₄are both an alkyl group; and R₅ is —H and R₆ is —H or methyl. Even morepreferably, R₁ and R₂ are both cyclopropyl or 1-methylcyclopropyl; R₃and R₄ are both an alkyl group, preferably methyl or ethyl; and R₅ is —Hand R₆ is —H or methyl.

The following are specific examples of bis(thio-hydrazide amide) disaltsrepresented by Structural Formula (II): R₁ and R₂ are both phenyl; R₃and R₄ are both methyl; R₅ is —H, and R₆ is ethyl; R₁ and R₂ are bothphenyl; R₃ and R₄ are both phenyl, and R₅ and R₆ are both methyl; R₁ andR₂ are both 2-thienyl; R₃ and R₄ are both phenyl, and R₅ and R₆ are bothmethyl; R₁ and R₂ are both 4-cyanophenyl; R₃ and R₄ are both methyl; R₅is —H, and R₆ is methyl; R₁ and R₂ are both phenyl; R₃ and R₄ are bothmethyl; R₅ is —H, and R₆ is methyl; R₁ and R₂ are both phenyl; R₃ and R₄are both methyl; R₅ is —H, and R₆ is benzyl; R₁ and R₂ are both phenyl;R₃ and R₄ are both methyl; R₅ is —H, and R₆ is ethyl; R₁ and R₂ are bothphenyl; R₃ and R₄ are both ethyl; R₅ is —H, and R₆ is n-butyl; R₁ and R₂are both 2,5-dimethoxyphenyl; R₃ and R₄ are both methyl; R₅ is —H, andR₆ is methyl; R₁ and R₂ are both phenyl; R₃ and R₄ are both methyl; R₅is —H, and R₆ is iso-propyl; R₁ and R₂ are both 3-nitrophenyl; R₃ and R₄are both methyl; R₅ is —H, and R₆ is methyl; R₁ and R₂ are both4-chlorophenyl; R₃ and R₄ are both methyl; R₅ is —H, and R₆ is methyl;R₁ and R₂ are both phenyl; R₃ and R₄ are both methyl; R₅ is —H, and R₆is 3-thienyl; R₁ and R₂ are both phenyl; R₃ and R₄ are both methyl, andR₅ and R₆, taken together, are propylene; R₁ and R₂ are both2,3-dimethoxyphenyl; R₃ and R₄ are both methyl; R₅ is —H, and R₆ ismethyl; R₁ and R₂ are both 2-chloro-5-methoxy phenyl; R₃ and R₄ are bothmethyl; R₅ is —H, and R₆ is methyl; R₁ and R₂ are both2,5-difluorophenyl; R₃ and R₄ are both methyl; R₅ is —H, and R₆ ismethyl; R₁ and R₂ are both 2,5-dichlorophenyl; R₃ and R₄ are bothmethyl; R₅ is —H, and R₆ is methyl; R₁ and R₂ are both2,6-dimethoxyphenyl; R₃ and R₄ are both methyl; R₅ is —H, and R₆ ismethyl; R₁ and R₂ are both 2,5-dimethylphenyl; R₃ and R₄ are bothmethyl; R₅ is —H, and R₆ is methyl; R₁ and R₂ are both2,5-dimethoxyphenyl; R₃ and R₄ are both ethyl; R₅ is —H, and R₆ ismethyl, and R₁ and R₂ are both 2,5-diethoxyphenyl; R₃ and R₄ are bothmethyl; R₅ is —H, and R₆ is methyl; R₁ and R₂ are both cyclopropyl; R₃and R₄ are both methyl; R₅ and R₆ are both —H; R₁ and R₂ are bothcyclopropyl; R₃ and R₄ are both ethyl; R₅ and R₆ are both —H; R and R₂are both cyclopropyl; R₃ and R₄ are both methyl; R₅ is methyl; R₆ is —H;R₁ and R₂ are both 1-methylcyclopropyl; R₃ and R₄ are both methyl; R₅and R₆ are both —H; R₁ and R₂ are both 1-methylcyclopropyl; R₃ and R₄are both methyl; R₅ is methyl and R₆ is —H; R₁ and R₂ are both1-methylcyclopropyl; R₃ and R₄ are both methyl; R₅ is ethyl and R₆ is—H; R₁ and R₂ are both 1-methylcyclopropyl; R₃ and R₄ are both methyl;R₅ is n-propyl and R₆ is —H; R₁ and R₂ are both 1-methylcyclopropyl; R₃and R₄ are both methyl; R₅ and R₆ are both methyl; R₁ and R₂ are both1-methylcyclopropyl; R₃ and R₄ are both ethyl; R₅ and R₆ are both —H; R₁and R₂ are both 1-methylcyclopropyl; R₃ is methyl, and R₄ is ethyl; R₅and R₆ are both —H; R₁ and R₂ are both 2-methylcyclopropyl; R₃ and R₄are both methyl; R₅ and R₆ are both —H; R₁ and R₂ are both2-phenylcyclopropyl; R₃ and R₄ are both methyl; R₅ and R₆ are both —H;R₁ and R₂ are both 1-phenylcyclopropyl; R₃ and R₄ are both methyl; R₅and R₆ are both —H; R₁ and R₂ are both cyclobutyl; R₃ and R₄ are bothmethyl; R₅ and R₆ are both —H; R₁ and R₂ are both cyclopentyl; R₃ and R₄are both methyl; R₅ and R₆ are both —H; R₁ and R₂ are both cyclohexyl;R₃ and R₄ are both methyl; R₅ and R₆ are both —H; R₁ and R₂ are bothcyclohexyl; R₃ and R₄ are both phenyl; R₅ and R₆ are both —H; R₁ and R₂are both methyl; R₃ and R₄ are both methyl; R₅ and R₆ are both —H; R₁and R₂ are both methyl; R₃ and R₄ are both t-butyl; R₅ and R₆ are both—H; R₁ and R₂ are both methyl; R₃ and R₄ are both phenyl; R₅ and R₆ areboth —H; R₁ and R₂ are both t-butyl; R₃ and R₄ are both methyl; R₅ andR₆ are both —H; R₁ and R₂ are ethyl; R₃ and R₄ are both methyl; R₅ andR₆ are both —H; R₁ and R₂ are both n-propyl; R₃ and R₄ are both methyl;R₅ and R₆ are both —H. In these examples, the pharmaceuticallyacceptable cation represented by M⁺ and M²⁺ is as described forStructural Formula (I), preferably Li⁺, Na⁺, K⁺, NH₃(C₂H₅OH)⁺ orN(CH₃)₃(C₂H₅OH)⁺, more preferably Na⁺ or K⁺, even more preferablyNa^(+.)

For many bis(thio-hydrazide amide) disalts represented by StructuralFormula (II), Y is —CH₂—. Examples include wherein: R₁ and R₂ are bothphenyl, and R₃ and R₄ are both o-CH₃-phenyl; R₁ and R₂ are botho—CH₃C(O)O-phenyl, and R₃ and R₄ are phenyl; R₁ and R₂ are both phenyl,and R₃ and R₄ are both methyl; R₁ and R₂ are both phenyl, and R₃ and R₄are both ethyl; R₁ and R₂ are both phenyl, and R₃ and R₄ are bothn-propyl; R₁ and R₂ are both p-cyanophenyl, and R₃ and R₄ are bothmethyl; R₁ and R₂ are both p-nitro phenyl, and R₃ and R₄ are bothmethyl; R₁ and R₂ are both 2,5-dimethoxyphenyl, and R₃ and R₄ are bothmethyl; R₁ and R₂ are both phenyl, and R₃ and R₄ are both n-butyl; R₁and R₂ are both p-chlorophenyl, and R₃ and R₄ are both methyl; R₁ and R₂are both 3-nitrophenyl, and R₃ and R₄ are both methyl; R₁ and R₂ areboth 3-cyanophenyl, and R₃ and R₄ are both methyl; R₁ and R₂ are both3-fluorophenyl, and R₃ and R₄ are both methyl; R₁ and R₂ are both2-furanyl, and R₃ and R₄ are both phenyl; R₁ and R₂ are both2-methoxyphenyl, and R₃ and R₄ are both methyl; R₁ and R₂ are both3-methoxyphenyl, and R₃ and R₄ are both methyl; R₁ and R₂ are both2,3-dimethoxyphenyl, and R₃ and R₄ are both methyl; R₁ and R₂ are both2-methoxy-5-chlorophenyl, and R₃ and R₄ are both ethyl; R₁ and R₂ areboth 2,5-difluorophenyl, and R₃ and R₄ are both methyl; R₁ and R₂ areboth 2,5-dichlorophenyl, and R₃ and R₄ are both methyl; R₁ and R₂ areboth 2,5-dimethylphenyl, and R₃ and R₄ are both methyl; R₁ and R₂ areboth 2-methoxy-5-chlorophenyl, and R₃ and R₄ are both methyl; R₁ and R₂are both 3,6-dimethoxyphenyl, and R₃ and R₄ are both methyl; R₁ and R₂are both phenyl, and R₃ and R₄ are both 2-ethylphenyl; R₁ and R₂ areboth 2-methyl-5-pyridyl, and R₃ and R₄ are both methyl; or R₁ is phenyl;R₂ is 2,5-dimethoxyphenyl, and R₃ and R₄ are both methyl; R₁ and R₂ areboth methyl, and R₃ and R₄ are both p-CF₃-phenyl; R₁ and R₂ are bothmethyl, and R₃ and R₄ are both o-CH₃-phenyl; R₁ and R₂ are both-CH₂)₃COOH; and R₃ and R₄ are both phenyl; R₁ and R₂ are bothrepresented by the following structural formula:

and R₃ and R₄ are both phenyl; R₁ and R₂ are both n-butyl, and R₃ and R₄are both phenyl; R₁ and R₂ are both n-pentyl, R₃ and R₄ are both phenyl;R₁ and R₂ are both methyl, and R₃ and R₄ are both 2-pyridyl; R₁ and R₂are both cyclohexyl, and R₃ and R₄ are both phenyl; R₁ and R₂ are bothmethyl, and R₃ and R₄ are both 2-ethylphenyl; R₁ and R₂ are both methyl,and R₃ and R₄ are both 2,6-dichlorophenyl; R₁-R₄ are all methyl; R₁ andR₂ are both methyl, and R₃ and R₄ are both t-butyl; R₁ and R₂ are bothethyl, and R₃ and R₄ are both methyl; R₁ and R₂ are both t-butyl, and R₃and R₄ are both methyl; R₁ and R₂ are both cyclopropyl, and R₃ and R₄are both methyl; R₁ and R₂ are both cyclopropyl, and R₃ and R₄ are bothethyl; R₁ and R₂ are both I-methylcyclopropyl, and R₃ and R₄ are bothmethyl; R₁ and R₂ are both 2-methylcyclopropyl, and R₃ and R₄ are bothmethyl; R₁ and R₂ are both 1-phenylcyclopropyl, and R₃ and R₄ are bothmethyl; R₁ and R₂ are both 2-phenylcyclopropyl, and R₃ and R₄ are bothmethyl; R₁ and R₂ are both cyclobutyl, and R₃ and R₄ are both methyl; R₁and R₂ are both cyclopentyl, and R₃ and R₄ are both methyl; R₁ iscyclopropyl, R₂ is phenyl, and R₃ and R₄ are both methyl. In theseexamples, the pharmaceutically acceptable cation represented by M⁺ andM²⁺ is as described for Structural Formula (I), preferably Li⁺, Na⁺, K⁺,NH₃(C₂H₅OH)⁺ or N(CH₃)₃(C₂H₅OH)⁺, more preferably Na⁺ or K⁺, even morepreferably Na⁺.

Preferred examples of bis(thio-hydrazide amide) disalts of the presentinvention are the following:

2 M⁺ and M²⁺ are as described above for Structural Formula (I).Preferably, the pharmaceutically acceptable cation is 2 M⁺, wherein M⁺is Li⁺, Na⁺, K⁺, NH₃(C₂H₅OH)⁺ or N(CH₃)₃(C₂H₅OH)⁺. More preferably, M⁺is Na⁺ or K⁺. Even more preferably, M₊ is Na⁺.

In Structural Formulas (I)-(II), R₁ and R₂ are the same or differentand/or R₃ and R₄ are the same or different. Preferably, R₁ and R₂ arethe same and R₃ and R₄ are the same.

The disclosed bis(thio-hydrazide amide) disalts may have tautomericforms. By way of example, tautomeric forms of the compounds representedby, for example, Structural Formula (II) wherein Y is —CH₂— are shownbelow:

It is to be understood when one tautomeric form of a disclosed compoundis depicted structurally, other tautomeric forms are also encompassed.

Certain compounds of the invention may be obtained as differentstereoisomers (e.g., diastereomers and enantiomers). The inventionincludes all isomeric forms and racemic mixtures of the disclosedcompounds and methods of treating a subject with both pure isomers andmixtures thereof, including racemic mixtures. Stereoisomers can beseparated and isolated using any suitable method, such aschromatography.

A “straight chained hydrocarbyl group” is an alkylene group, i.e.,—(CH₂)_(y)—, with one or more (preferably one) internal methylene groupsoptionally replaced with a linkage group. y is a positive integer (e.g.,between 1 and 10), preferably between 1 and 6 and more preferably 1 or2. A “linkage group” refers to a functional group which replaces amethylene in a straight chained hydrocarbyl. Examples of suitablelinkage groups include a ketone (—C(O)—), alkene, alkyne, phenylene,ether (—O—), thioether (—S—), or amine (—N(R^(a))—), wherein R^(a) isdefined below. A preferred linkage group is —C(R₅R₆)—, wherein R₅ and R₆are defined above. Suitable substitutents for an alkylene group and ahydrocarbyl group are those which do not substantially interfere withthe anti-cancer activity of the disclosed compounds. R₅ and R₆ arepreferred substituents for an alkylene or hydrocarbyl group representedby Y.

An aliphatic group is a straight chained, branched or cyclicnon-aromatic hydrocarbon which is completely saturated or which containsone or more units of unsaturation. Typically, a straight chained orbranched aliphatic group has from 1 to about 20 carbon atoms, preferablyfrom 1 to about 10, and a cyclic aliphatic group has from 3 to about 10carbon atoms, preferably from 3 to about 8. An aliphatic group ispreferably a straight chained or branched alkyl group, e.g, methyl,ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, tert-butyl, pentyl,hexyl, pentyl or octyl, or a cycloalkyl group with 3 to about 8 carbonatoms. A C1-C8 straight chained or branched alkyl group or a C3-C8cyclic alkyl group is also referred to as a “lower alkyl” group.

The term “aromatic group” may be used interchangeably with “aryl,” “arylring,” “aromatic ring,” “aryl group” and “aromatic group.” Aromaticgroups include carbocyclic aromatic groups such as phenyl, naphthyl, andanthracyl, and heteroaryl groups such as imidazolyl, thienyl, furanyl,pyridyl, pyrimidy, pyranyl, pyrazolyl, pyrroyl, pyrazinyl, thiazole,oxazolyl, and tetrazole. The term “heteroaryl group” may be usedinterchangeably with “heteroaryl,” “heteroaryl ring,” “heteroaromaticring” and “heteroaromatic group.” Heteroaryl groups are aromatic groupsthat comprise one or more heteroatom, such as sulfur, oxygen andnitrogen, in the ring structure. Preferably, heteroaryl groups comprisefrom one to four heteroatoms.

Aromatic groups also include fused polycyclic aromatic ring systems inwhich a carbocyclic aromatic ring or heteroaryl ring is fused to one ormore other heteroaryl rings. Examples include benzothienyl,benzofuranyl, indolyl, quinolinyl, benzothiazole, benzooxazole,benzimidazole, quinolinyl, isoquinolinyl and isoindolyl.

The term “arylene” refers to an aryl group which is connected to theremainder of the molecule by two other bonds. By way of example, thestructure of a 1,4-phenylene group is shown below:

Substituents for an arylene group are as described below for an arylgroup.

Non-aromatic heterocyclic rings are non-aromatic rings which include oneor more heteroatoms such as nitrogen, oxygen or sulfur in the ring. Thering can be five, six, seven or eight-membered. Preferably, heterocyclicgroups comprise from one to about four heteroatoms. Examples includetetrahydrofuranyl, tetrahyrothiophenyl, morpholino, thiomorpholino,pyrrolidinyl, piperazinyl, piperidinyl, and thiazolidinyl.

Suitable substituents on an aliphatic group (including an alkylenegroup), non-aromatic heterocyclic group, benzylic or aryl group(carbocyclic and heteroaryl) are those which do not substantiallyinterfere with the anti-cancer activity of the disclosed compounds. Asubstituent substantially interferes with anti-cancer activity when theanti-cancer activity is reduced by more than about 50% in a compoundwith the substituent compared with a compound without the substituent.Examples of suitable substituents include —R^(a), —OH, —Br, —Cl, I, —F,—OR^(a), —O—COR^(a), —COR^(a), —CN, —NO₂, —COOH, —SO₃H, —NH₂, —NHR^(a),—N(R^(a)R^(b)), —COOR^(a), —CHO, —CONH₂, —CONHR^(a), —CON(R^(a)R^(b)),—NHCOR^(a), —NR^(c)COR^(a), —NHCONH₂, —NHCONR^(a)H, —NHCON(R^(a)R^(b)),—NR^(c)CONH₂, —NR^(c)CONR^(a)H, —NR^(c)CON(R^(a)R^(b)), —C(═NH)—NH₂,—C(═NH)—NHR^(a), —C(═NH)—N(R^(a)R^(b)), —C(═NR^(c))—NH₂,—C(═NR^(c))—NHR^(a), —C(═NR^(c))—N(R^(a)R^(b)), —NH—C(═NH)—NH₂,—NH—C(═NH)—NHR^(a), —NH—C(═NH)—N(R^(a)R^(b)), —NH—C(═NR^(c))—NH₂,—NH—C(═NR^(c))—NHR^(a), —NH—C(═NR^(c))—N(R^(a)R^(b)),—NR^(d)H—C(═NH)—NH₂, —NR^(d)—C(═NH)—NHR^(a),—NR^(d)—C(═NH)—N(R^(a)R^(b)), —NR^(d)—C(═NR^(c))—NH₂,—NR^(d)—C(═NR^(c))—NHR^(a), —NR^(d)—C(═NR^(c))—N(R^(a)R^(b)), —NHNH₂,—NHNHR^(a), —NHR^(a)R^(b), —SO₂NH₂, —SO₂NHR^(a), —SO₂NR^(a)R^(b),—CH═CHR^(a), —CH═CR^(a)R^(b), —CR^(c)═CR^(a)R^(b),—CR^(c)═CHR^(a),—CR^(c)═CR^(a)R^(b), —CCR^(a), —SH, —SR^(a), —S(O)R^(a), —S(O)₂R^(a).

R^(a)—R^(d) are each independently an alkyl group, aromatic group,non-aromatic heterocyclic group or —N(R^(a)R^(b)), taken together, forma substituted or unsubstituted non-aromatic heterocyclic group. Thealkyl, aromatic and non-aromatic heterocyclic group represented byR^(a)—R^(d) and the non-aromatic heterocyclic group represented by—N(R^(a)R^(b)) are each optionally and independently substituted withone or more groups represented by R^(#).

R^(#) is R⁺, —OR⁺, —O(haloalkyl), —SR⁺, —NO₂, —CN, —NCS, —N(R⁺)₂,—NHCO₂R⁺, —NHC(O)R⁺, —NHNHC(O)R⁺, —NHC(O)N(R⁺)₂, —NHNHC(O)N(R⁺)₂,—NHNHCO₂R⁺, —C(O)C(O)R⁺, —C(O)CH₂C(O)R⁺, —CO₂R⁺, —C(O)R⁺, —C(O)N(R⁺)₂,—OC(O)R⁺, —OC(O)N(R⁺)₂, —S(O)₂R⁺, —SO₂N(R⁺)₂, —S(O)R⁺, —NHSO₂N(R⁺)₂,—NHSO₂R⁺, —C(═S)N(R⁺)₂, or —C(═NH)—N(R⁺)₂.

R⁺is —H, a C1-C4 alkyl group, a monocyclic heteroaryl group, anon-aromatic heterocyclic group or a phenyl group optionally substitutedwith alkyl, haloalkyl, alkoxy, haloalkoxy, halo, —CN, —NO₂, amine,alkylamine or dialkylamine. Optionally, the group —N(R⁺)₂ is anon-aromatic heterocyclic group, provided that non-aromatic heterocyclicgroups represented by R⁺and —N(R⁺)₂ that comprise a secondary ring amineare optionally acylated or alkylated.

Preferred substituents for a phenyl group, including phenyl groupsrepresented by R₁-R₄, include C1-C4 alkyl, C1-C4 alkoxy, C1-C4haloalkyl, C1-C4 haloalkoxy, phenyl, benzyl, pyridyl, —OH, —NH₂, —F,—Cl, —Br, —I, —NO₂ or —CN.

Preferred substituents for a cycloalkyl group, including cycloalkylgroups represented by R₁ and R₂, are alkyl groups, such as a methyl orethyl group.

Another embodiment of the present invention is a pharmaceuticalcomposition comprising a bis(thio-hydrazide amide) disalt disclosedherein and a pharmaceutically acceptable carrier or diluent.

Suitable pharmaceutically acceptable carriers may contain inertingredients which do not inhibit the biological activity of thedisclosed disalts. The pharmaceutically acceptable carriers should bebiocompatible, i.e., non-toxic, non-inflammatory, non-immunogenic anddevoid of other undesired reactions upon the administration to asubject. Standard pharmaceutical formulation techniques can be employed,such as those described in Remington's Pharmaceutical Sciences, MackPublishing Company, Easton, Pa. Formulation of the compound to beadministered will vary according to the route of administration selected(e.g., solution, emulsion, capsule). Suitable pharmaceutical carriersfor parenteral administration include, for example, sterile water,physiological saline, bacteriostatic saline (saline containing about0.9% mg/ml benzyl alcohol), phosphate-buffered saline, Hank's solution,Ringer's-lactate and the like. Methods for encapsulating compositions(such as in a coating of hard gelatin or cyclodextrins) are known in theart (Baker, et al., “Controlled Release of Biological Active Agents”,John Wiley and Sons, 1986).

Another embodiment of the present invention is a method of treating asubject with a cancer. Optionally, the method of the invention can beused for a multi-drug resistant cancer as described below. The methodcomprises the step of administering an effective amount of abis(thio-hydrazide amide) disalt described herein. Preferably, one ormore additional anti-cancer drugs are co-administered with thebis(thio-hydrazide amide) disalt. Examples of anti-cancer drugs aredescribed below. Preferably, the co-administered anti-cancer drug is anagent that stabilizes mictotubules, such as paclitaxel or an analog ofpaclitaxel.

A “subject” is a mammal, preferably a human, but can also be an animalin need of veterinary treatment, e.g., companion animals (e.g., dogs,cats, and the like), farm animals (e.g., cows, sheep, pigs, horses, andthe like) and laboratory animals (e.g., rats, mice, guinea pigs, and thelike).

As noted above, one embodiment of the present invention is directed totreating subjects with a cancer. “Treating a subject with a cancer”includes achieving, partially or substantially, one or more of thefollowing: arresting the growth or spread of a cancer, reducing theextent of a cancer (e.g., reducing size of a tumor or reducing thenumber of affected sites), inhibiting the growth rate of a cancer, andameliorating or improving a clinical symptom or indicator associatedwith a cancer (such as tissue or serum components).

Cancers that can be treated or prevented by the methods of the presentinvention include, but are not limited to human sarcomas and carcinomas,e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma,retinoblastoma; leukemias, e.g., acute lymphocytic leukemia and acutemyelocytic leukemia (myeloblastic, promyelocytic, myelomonocytic,monocytic and erythroleukemia); chronic leukemia (chronic myelocytic(granulocytic) leukemia and chronic lymphocytic leukemia); andpolycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin'sdisease), multiple myeloma, Waldenstrobm's macroglobulinemia, and heavychain disease.

Other examples of leukemias include acute and/or chronic leukemias,e.g., lymphocytic leukemia (e.g., as exemplified by the p388 (murine)cell line), large granular lymphocytic leukemia, and lymphoblasticleukemia; T-cell leukemias, e.g., T-cell leukemia (e.g., as exemplifiedby the CEM, Jurkat, and HSB-2 (acute), YAC-1(murine) cell lines),T-lymphocytic leukemia, and T-lymphoblastic leukemia; B cell leukemia(e.g., as exemplified by the SB (acute) cell line), and B-lymphocyticleukemia; mixed cell leukemias, e.g., B and T cell leukemia and B and Tlymphocytic leukemia; myeloid leukemias, e.g., granulocytic leukemia,myelocytic leukemia (e.g., as exemplified by the HL-60 (promyelocyte)cell line), and myelogenous leukemia (e.g., as exemplified by theK562(chronic)cell line); neutrophilic leukemia; eosinophilic leukemia;monocytic leukemia (e.g., as exemplified by the THP-1(acute) cell line);myelomonocytic leukemia; Naegeli-type myeloid leukemia; andnonlymphocytic leukemia. Other examples of leukemias are described inChapter 60 of The Chemotherapy Sourcebook, Michael C. Perry Ed.,Williams & Williams (1992) and Section 36 of Holland Frie CancerMedicine 5th Ed., Bast et al. Eds., B.C. Decker Inc. (2000). The entireteachings of the preceding references are incorporated herein byreference.

In one embodiment, the disclosed method is believed to be particularlyeffective in treating a subject with non-solid tumors such as multiplemyeloma. In another embodiment, the disclosed method is believed to beparticularly effective against T-leukemia (e.g., as exemplified byJurkat and CEM cell lines); B-leukemia (e.g., as exemplified by the SBcell line); promyelocytes (e.g., as exemplified by the HL-60 cell line);uterine sarcoma (e.g., as exemplified by the MES-SA cell line);monocytic leukemia (e.g., as exemplified by the THP-1(acute) cell line);and lymphoma (e.g., as exemplified by the U937 cell line); mostpreferably, this embodiment of the method employs the disodium salt ofCompound (1).

The disclosed method is particularly effective at treating subjectswhose cancer has become “multi-drug resistant”. A cancer which initiallyresponded to an anti-cancer drug becomes resistant to the anti-cancerdrug when the anti-cancer drug is no longer effective in treating thesubject with the cancer. For example, many tumors will initially respondto treatment with an anti-cancer drug by decreasing in size or evengoing into remission, only to develop resistance to the drug. Drugresistant tumors are characterized by a resumption of their growthand/or reappearance after having seemingly gone into remission, despitethe administration of increased dosages of the anti-cancer drug. Cancersthat have developed resistance to two or more anti-cancer drugs are saidto be “multi-drug resistant”. For example, it is common for cancers tobecome resistant to three or more anti-cancer agents, often five or moreanti-cancer agents and at times ten or more anti-cancer agents.

An “effective amount” is the quantity of compound in which a beneficialclinical outcome is achieved when the compound is administered to asubject with a cancer. A “beneficial clinical outcome” includes areduction in tumor mass, a reduction in metastasis, a reduction in theseverity of the symptoms associated with the cancer and/or an increasein the longevity of the subject compared with the absence of thetreatment. The precise amount of compound administered to a subject willdepend on the type and severity of the disease or condition and on thecharacteristics of the subject, such as general health, age, sex, bodyweight and tolerance to drugs. It will also depend on the degree,severity and type of cancer. The skilled artisan will be able todetermine appropriate dosages depending on these and other factors.Effective amounts of the disclosed compounds typically range betweenabout 1 mg/mm² per day and about 10 grams/mm² per day, and preferablybetween 10 mg/mm² per day and about 5 grams/mm². When co-administeredwith another anti-cancer agent, an “effective amount” of the secondanti-cancer agent will depend on the type of drug used. Suitable dosagesare known for approved anti-cancer agents and can be adjusted by theskilled artisan according to the condition of the subject, the type ofcancer being treated and the amount of bis(thio-hydrazide amide) disaltbeing used.

The disclosed bis(thio-hydrazide amide) disalts are administered by anysuitable route, including, for example, orally in capsules, suspensionsor tablets or by parenteral administration. Parenteral administrationcan include, for example, systemic administration, such as byintramuscular, intravenous, subcutaneous, or intraperitoneal injection.The bis(thio-hydrazide amide) disalts can also be administered orally(e.g., dietary), topically, by inhalation (e.g., intrabronchial,intranasal, oral inhalation or intranasal drops), or rectally, dependingon the type of cancer to be treated. Oral and parenteral administrationsare preferred modes of administration.

Optionally, the disclosed bis(thio-hydrazide amide) disalts can beco-administered with other anti-cancer agents such as Adriamycin,Dactinomycin, Bleomycin, Vinblastine, Cisplatin, acivicin; aclarubicin;acodazole hydrochloride; acronine; adozelesin; aldesleukin; altretamine;ambomycin; ametantrone acetate; aminoglutethimide; amsacrine;anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa;azotomycin; batimastat; benzodepa; bicalutamide; bisantrenehydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate;brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone;caracemide; carbetimer; carboplatin; carmustine; carubicinhydrochloride; carzelesin; cedefingol; chlorambucil; cirolemycin;cladribine; crisnatol mesylate; cyclophosphamide; cytarabine;dacarbazine; daunorubicin hydrochloride; decitabine; dexormaplatin;dezaguanine; dezaguanine mesylate; diaziquone; doxorubicin; doxorubicinhydrochloride; droloxifene; droloxifene citrate; dromostanolonepropionate; duazomycin; edatrexate; eflomithine hydrochloride;elsamitrucin; enloplatin; enpromate; epipropidine; epirubicinhydrochloride; erbulozole; esorubicin hydrochloride; estramustine;estramustine phosphate sodium; etanidazole; etoposide; etoposidephosphate; etoprine; fadrozole hydrochloride; fazarabine; fenretinide;floxuridine; fludarabine phosphate; fluorouracil; flurocitabine;fosquidone; fostriecin sodium; gemcitabine; gemcitabine hydrochloride;hydroxyurea; idarubicin hydrochloride; ifosfamide; ilmofosine;interleukin II (including recombinant interleukin II, or rIL2),interferon alfa-2a; interferon alfa-2b; interferon alfa-n1; interferonalfa-n3; interferon beta-I a; interferon gamma-I b; iproplatin;irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolideacetate; liarozole hydrochloride; lometrexol sodium; lomustine;losoxantrone hydrochloride; masoprocol; maytansine; mechlorethaminehydrochloride; megestrol acetate; melengestrol acetate; melphalan;menogaril; mercaptopurine; methotrexate; methotrexate sodium; metoprine;meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin; mitomalcin;mitomycin; mitosper; mitotane; mitoxantrone hydrochloride; mycophenolicacid; nocodazole; nogalamycin; ormaplatin; oxisuran; pegaspargase;peliomycin; pentamustine; peplomycin sulfate; perfosfamide; pipobroman;piposulfan; piroxantrone hydrochloride; plicamycin; plomestane; porfimersodium; porfiromycin; prednimustine; procarbazine hydrochloride;puromycin; puromycin hydrochloride; pyrazofurin; riboprine; rogletimide;safingol; safingol hydrochloride; semustine; simtrazene; sparfosatesodium; sparsomycin; spirogermanium hydrochloride; spiromustine;spiroplatin; streptonigrin; streptozocin; sulofenur; talisomycin;tecogalan sodium; tegafur; teloxantrone hydrochloride; temoporfin;teniposide; teroxirone; testolactone; thiamiprine; thioguanine;thiotepa; tiazofurin; tirapazamine; toremifene citrate; trestoloneacetate; triciribine phosphate; trimetrexate; trimetrexate glucuronate;triptorelin; tubulozole hydrochloride; uracil mustard; uredepa;vapreotide; verteporfin; vinblastine sulfate; vincristine sulfate;vindesine; vindesine sulfate; vinepidine sulfate; vinglycinate sulfate;vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate;vinzolidine sulfate; vorozole; zeniplatin; zinostatin; zorubicinhydrochloride.

Other anti-cancer drugs include, but are not limited to: 20-epi-1,25dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin;acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK antagonists;altretamine; ambamustine; amidox; amifostine; aminolevulinic acid;amrubicin; amsacrine; anagrelide; anastrozole; andrographolide;angiogenesis inhibitors; antagonist D; antagonist G; antarelix;anti-dorsalizing morphogenetic protein-1; antiandrogen, prostaticcarcinoma; antiestrogen; antineoplaston; antisense oligonucleotides;aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators;apurinic acid; ara-CDP-DL-PTBA; arginine deaminase; asulacrine;atamestane; atrimustine; axinastatin 1; axinastatin 2; axinastatin 3;azasetron; azatoxin; azatyrosine; baccatin III derivatives; balanol;batimastat; BCR/ABL antagonists; benzochlorins; benzoylstaurosporine;beta lactam derivatives; beta-alethine; betaclamycin B; betulinic acid;bFGF inhibitor; bicalutamide; bisantrene; bisaziridinylspermine;bisnafide; bistratene A; bizelesin; breflate; bropirimine; budotitane;buthionine sulfoximine; calcipotriol; calphostin C; camptothecinderivatives; canarypox IL-2; capecitabine; carboxamide-amino-triazole;carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor;carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropinB; cetrorelix; chlorlns; chloroquinoxaline sulfonamide; cicaprost;cis-porphyrin; cladribine; clomifene analogues; clotrimazole;collismycin A; collismycin B; combretastatin A4; combretastatinanalogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8;cryptophycin A derivatives; curacin A; cyclopentanthraquinones;cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor;cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin;dexamethasone; dexifosfamide; dexrazoxane; dexverapamil; diaziquone;didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine;9-dioxamycin; diphenyl spiromustine; docosanol; dolasetron;doxifluridine; droloxifene; dronabinol; duocarmycin SA; ebselen;ecomustine; edelfosine; edrecolomab; eflomithine; elemene; emitefur;epirubicin; epristeride; estramustine analogue; estrogen agonists;estrogen antagonists; etanidazole; etoposide phosphate; exemestane;fadrozole; fazarabine; fenretinide; filgrastim; finasteride;flavopiridol; flezelastine; fluasterone; fludarabine; fluorodaunorunicinhydrochloride; forfenimex; formestane; fostriecin; fotemustine;gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix;gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam;heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid;idarubicin; idoxifene; idramantone; ilmofosine; ilomastat;imidazoacridones; imiquimod; immunostimulant peptides; insulin-likegrowth factor-1 receptor inhibitor; interferon agonists; interferons;interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-; iroplact;irsogladine; isobengazole; isohomohalicondrin B; itasetron;jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide;leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole;leukemia inhibiting factor; leukocyte alpha interferon;leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole;linear polyamine analogue; lipophilic disaccharide peptide; lipophilicplatinum compounds; lissoclinamide 7; lobaplatin; lombricine;lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine;lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides;maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysininhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone;meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone;miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone;mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growthfactor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonalantibody, human chorionic gonadotrophin; monophosphoryl lipidA+myobacterium cell wall sk; mopidamol; multiple drug resistance geneinhibitor; multiple tumor suppressor 1-based therapy; mustard anticanceragent; mycaperoxide B; mycobacterial cell wall extract; myriaporone;N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip;naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin;nemorubicin; neridronic acid; neutral endopeptidase; nilutamide;nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn;O6-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone;ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin;osaterone; oxaliplatin; oxaunomycin; palauamine; palmitoylrhizoxin;pamidronic acid; panaxytriol; panomifene; parabactin; pazelliptine;pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin;pentrozole; perflubron; perfosfamide; perillyl alcohol; phenazinomycin;phenylacetate; phosphatase inhibitors; picibanil; pilocarpinehydrochloride; pirarubicin; piritrexim; placetin A; placetin B;plasminogen activator inhibitor; platinum complex; platinum compounds;platinum-triamine complex; porfimer sodium; porfiromycin; prednisone;propyl bis-acridone; prostaglandin J2; proteasome inhibitors; proteinA-based immune modulator; protein kinase C inhibitor; protein kinase Cinhibitors, microalgal; protein tyrosine phosphatase inhibitors; purinenucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine;pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists;raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors;ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide;rohitukine; romurtide; roquinimex; rubiginone B1; ruboxyl; safingol;saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics;semustine; senescence derived inhibitor 1; sense oligonucleotides;signal transduction inhibitors; signal transduction modulators; singlechain antigen-binding protein; sizofiran; sobuzoxane; sodiumborocaptate; sodium phenylacetate; solverol; somatomedin bindingprotein; sonermin; sparfosic acid; spicamycin D; spiromustine;splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem-celldivision inhibitors; stipiamide; stromelysin inhibitors; sulfinosine;superactive vasoactive intestinal peptide antagonist; suradista;suramin; swainsonine; synthetic glycosaminoglycans; tallimustine;tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium;tegafur; tellurapyrylium; telomerase inhibitors; temoporfin;temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine;thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic;thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroidstimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocenebichloride; topsentin; toremifene; totipotent stem cell factor;translation inhibitors; tretinoin; triacetyluridine; triciribine;trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinaseinhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenitalsinus-derived growth inhibitory factor; urokinase receptor antagonists;vapreotide; variolin B; vector system, erythrocyte gene therapy;velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine;vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; and zinostatinstimalamer. Preferred additional anti-cancer drugs are 5-fluorouraciland leucovorin.

Examples of therapeutic antibodies that can be used include but are notlimited to HERCEPTIN® (Trastuzumab) (Genentech, CA) which is a humanizedanti-HER2 monoclonal antibody for the treatment of patients withmetastatic breast cancer; REOPRO® (abciximab) (Centocor) which is ananti-glycoprotein IIb/IIIa receptor on the platelets for the preventionof clot formation; ZENAPAX® (daclizumab) (Roche Pharmaceuticals,Switzerland) which is an immunosuppressive, humanized anti-CD25monoclonal antibody for the prevention of acute renal allograftrejection; PANOREX™ which is a murine anti-17-IA cell surface antigenIgG2a antibody (Glaxo Wellcome/Centocor); BEC2 which is a murineanti-idiotype (GD3 epitope) IgG antibody (ImClone System); IMC-C225which is a chimeric anti-EGFR IgG antibody (ImClone System); VITAXIN™which is a humanized anti-αVβ3 integrin antibody (Applied MolecularEvolution/MedImmune); Campath 1H/LDP-03 which is a humanized anti CD52IgG1 antibody (Leukosite); Smart M195 which is a humanized anti-CD33 IgGantibody (Protein Design Lab/Kanebo); RITUXAN™ which is a chimericanti-CD20 IgG1 antibody (IDEC Pharm/Genentech, Roche/Zettyaku);LYMPHOCIDE™ which is a humanized anti-CD22 IgG antibody (Immunomedics);LYMPHOCIDE™ Y-90 (Immunomedics); Lymphoscan (Tc-99m-labeled;radioimaging; Immunomedics); Nuvion (against CD3; Protein Design Labs);CM3 is a humanized anti-ICAM3 antibody (ICOS Pharm); IDEC-114 is aprimatied anti-CD80 antibody (IDEC Pharm/Mitsubishi); ZEVALIN™ is aradiolabelled murine anti-CD20 antibody (IDEC/Schering AG); IDEC-131 isa humanized anti-CD40L antibody (IDEC/Eisai); IDEC-151 is a primatizedanti-CD4 antibody (IDEC); IDEC-152 is a primatized anti-CD23 antibody(IDEC/Seikagaku); SMART anti-CD3 is a humanized anti-CD3 IgG (ProteinDesign Lab); 5G1.1 is a humanized anti-complement factor 5 (C5) antibody(Alexion Pharm); D2E7 is a humanized anti-TNF-α antibody (CAT/BASF);CDP870 is a humanized anti-TNF-α Fab fragment (Celltech); IDEC-151 is aprimatized anti-CD4 IgG1 antibody (IDEC Pharm/SmithKline Beecham);MDX-CD4 is a human anti-CD4 IgG antibody (Medarex/Eisai/Genmab);CD20-sreptdavidin (+biotin-yttrium 90; NeoRx); CDP571 is a humanizedanti-TNF-α IgG4 antibody (Celltech); LDP-02 is a humanized anti-α4β7antibody (LeukoSite/Genentech); OrthoClone OKT4A is a humanized anti-CD4IgG antibody (Ortho Biotech); ANTOVA™ is a humanized anti-CD40L IgGantibody (Biogen); ANTEGREN™ is a humanized anti-VLA-4 IgG antibody(Elan); and CAT-152 is a human anti-TGF-β2 antibody (Cambridge Ab Tech).

Chemotherapeutic agents that can be used in the methods and compositionsof the invention include but are not limited to alkylating agents,antimetabolites, natural products, or hormones. Examples of alkylatingagents useful for the treatment or prevention of T-cell malignancies inthe methods and compositions of the invention include but are notlimited to, nitrogen mustards (e.g., mechloroethamine, cyclophosphamide,chlorambucil, etc.), alkyl sulfonates (e.g., busulfan), nitrosoureas(e.g., carmustine, lomusitne, etc.), or triazenes (decarbazine, etc.).Examples of antimetabolites useful for the treatment or prevention ofT-cell malignancies in the methods and compositions of the inventioninclude but are not limited to folic acid analog (e.g., methotrexate),or pyrimidine analogs (e.g., Cytarabine), purine analogs (e.g.,mercaptopurine, thioguanine, pentostatin). Examples of natural productsuseful for the treatment or prevention of T-cell malignancies in themethods and compositions of the invention include but are not limited tovinca alkaloids (e.g., vinblastin, vincristine), epipodophyllotoxins(e.g., etoposide), antibiotics (e.g., daunorubicin, doxorubicin,bleomycin), enzymes (e.g., L-asparaginase), or biological responsemodifiers (e.g., interferon alpha).

Examples of alkylating agents useful for the treatment or prevention ofcancer in the methods and compositions of the invention include but arenot limited to, nitrogen mustards (e.g., mechloroethamine,cyclophosphamide, chlorambucil, melphalan, etc.), ethylenimine andmethylmelamines (e.g., hexamethlymelamine, thiotepa), alkyl sulfonates(e.g., busulfan), nitrosoureas (e.g., carmustine, lomusitne, semustine,streptozocin, etc.), or triazenes (decarbazine, etc.). Examples ofantimetabolites useful for the treatment or prevention of cancer in themethods and compositions of the invention include but are not limited tofolic acid analog (e.g., methotrexate), or pyrimidine analogs (e.g.,fluorouracil, floxouridine, Cytarabine), purine analogs (e.g.,mercaptopurine, thioguanine, pentostatin). Examples of natural productsuseful for the treatment or prevention of cancer in the methods andcompositions of the invention include but are not limited to vincaalkaloids (e.g., vinblastin, vincristine), epipodophyllotoxins (e.g.,etoposide, teniposide), antibiotics (e.g., actinomycin D, daunorubicin,doxorubicin, bleomycin, plicamycin, mitomycin), enzymes (e.g.,L-asparaginase), or biological response modifiers (e.g., interferonalpha). Examples of hormones and antagonists useful for the treatment orprevention of cancer in the methods and compositions of the inventioninclude but are not limited to adrenocorticosteroids (e.g., prednisone),progestins (e.g., hydroxyprogesterone caproate, megestrol acetate,medroxyprogesterone acetate), estrogens (e.g., diethlystilbestrol,ethinyl estradiol), antiestrogen (e.g., tamoxifen), androgens (e.g.,testosterone propionate, fluoxymesterone), antiandrogen (e.g.,flutamide), gonadotropin releasing hormone analog (e.g., leuprolide).Other agents that can be used in the methods and with the compositionsof the invention for the treatment or prevention of cancer includeplatinum coordination complexes (e.g., cisplatin, carboblatin),anthracenedione (e.g., mitoxantrone), substituted urea (e.g.,hydroxyurea), methyl hydrazine derivative (e.g., procarbazine),adrenocortical suppressant (e.g., mitotane, aminoglutethimide).

The bis(thio-hydrazide amide) disalts disclosed herein are believed tobe particularly effective when co-administered with anti-cancer agentswhich act by arresting cells in the G2-M phases due to stabilization ofmicrotubules. Thus, the disclosed method preferably includesco-administration of anti-cancer drugs which act by this mechanism.Examples of anti-cancer agents which act by arresting cells in the G2-Mphases due to stabilization of microtubules include without limitationthe following marketed drugs and drugs in development: Erbulozole (alsoknown as R-55104), Dolastatin 10 (also known as DLS-10 and NSC-376128),Mivobulin isethionate (also known as CI-980), Vincristine, NSC-639829,Discodermolide (also known as NVP-XX-A-296), ABT-751 (Abbott, also knownas E-7010), Altorhyrtins (such as Altorhyrtin A and Altorhyrtin C),Spongistatins (such as Spongistatin 1, Spongistatin 2, Spongistatin 3,Spongistatin 4, Spongistatin 5, Spongistatin 6, Spongistatin 7,Spongistatin 8, and Spongistatin 9), Cemadotin hydrochloride (also knownas LU-103793 and NSC-D-669356), Epothilones (such as Epothilone A,Epothilone B, Epothilone C (also known as desoxyepothilone A or dEpoA),Epothilone D (also referred to as KOS-862, dEpoB, and desoxyepothilone B), Epothilone E, Epothilone F, Epothilone B N-oxide, Epothilone AN-oxide, 16-aza-epothilone B, 21-aminoepothilone B (also known asBMS-310705), 21-hydroxyepothilone D (also known as Desoxyepothilone Fand dEpoF), 26-fluoroepothilone), Auristatin PE (also known asNSC-654663), Soblidotin (also known as TZT-1027), LS-4559-P (Pharmacia,also known as LS-4577), LS-4578 (Pharmacia, also known as LS-477-P),LS-4477 (Pharmacia), LS-4559 (Pharmacia), RPR-112378 (Aventis),Vincristine sulfate, DZ-3358 (Daiichi), FR-182877 (Fujisawa, also knownas WS-9885B), GS-164 (Takeda), GS-198 (Takeda), KAR-2 (Hungarian Academyof Sciences), BSF-223651 (BASF, also known as ILX-651 and LU-223651),SAH-49960 (Lilly/Novartis), SDZ-268970 (Lilly/Novartis), AM-97(Armad/Kyowa Hakko), AM-132 (Armad), AM-138 (Armad/Kyowa Hakko),IDN-5005 (Indena), Cryptophycin 52 (also known as LY-355703), AC-7739(Ajinomoto, also known as AVE-8063A and CS-39.HCl), AC-7700 (Ajinomoto,also known as AVE-8062, AVE-8062A, CS-39-L-Ser.HCl, and RPR-258062A),Vitilevuamide, Tubulysin A, Canadensol, Centaureidin (also known asNSC-106969), T-138067 (Tularik, also known as T-67, TL-138067 andTI-138067), COBRA-1 (Parker Hughes Institute, also known as DDE-261 andWHI-261), H10 (Kansas State University), H16 (Kansas State University),Oncocidin A1 (also known as BTO-956 and DIME), DDE-313 (Parker HughesInstitute), Fijianolide B, Laulimalide, SPA-2 (Parker Hughes Institute),SPA-1 (Parker Hughes Institute, also known as SPIKET-P), 3-IAABU(Cytoskeleton/Mt. Sinai School of Medicine, also known as MF-569),Narcosine (also known as NSC-5366), Nascapine, D-24851 (Asta Medica),A-105972 (Abbott), Hemiasterlin, 3-BAABU (Cytoskeleton/Mt. Sinai Schoolof Medicine, also known as MF-191), TMPN (Arizona State University),Vanadocene acetylacetonate, T-138026 (Tularik), Monsatrol, Inanocine(also known as NSC-698666), 3-IAABE (Cytoskeleton/Mt. Sinai School ofMedicine), A-204197 (Abbott), T-607 (Tularik, also known as T-900607),RPR-115781 (Aventis), Eleutherobins (such as Desmethyleleutherobin,Desaetyleleutherobin, Isoeleutherobin A, and Z-Eleutherobin),Caribaeoside, Caribaeolin, Halichondrin B, D-64131 (Asta Medica),D-68144 (Asta Medica), Diazonamide A, A-293620 (Abbott), NPI-2350(Nereus), Taccalonolide A, TUB-245 (Aventis), A-259754 (Abbott),Diozostatin, (−)-Phenylahistin (also known as NSCL-96F037), D-68838(Asta Medica), D-68836 (Asta Medica), Myoseverin B, D-43411 (Zentaris,also known as D-81862), A-289099 (Abbott), A-318315 (Abbott), HTI-286(also known as SPA-110, trifluoroacetate salt) (Wyeth), D-82317(Zentaris), D-82318 (Zentaris), SC-12983 (NCI), Resverastatin phosphatesodium, BPR-0Y-007 (National Health Research Institutes), and SSR-250411(Sanofi), and Hsp90 inhibitors such as geldanmycin, radicicol,herbimycin A, macbecin I and II, novobiocin,17-Allylamino-17-demethoxygeldanamycin (17AAG),17-Demethoxy-17-[2-(dimethylamino)ethylamino]geldanamycin (17DMAG),CNF-1010, purine-based Hsp90 inhibitors such as PU3, PU24FCl, andPU29FCl, and oxime derivatives of radicicol such as KF25706 and KF58333.

Paclitaxel, also referred to as “TAXOL”, is a well-known anti-cancerdrug which acts by enhancing and stabilizing microtubule formation. Thestructure of paclitaxel is shown in FIG. 1. Many analogs of paclitaxelare known, including Docetaxel, the structure of which is shown in FIG.2. Docetaxel is also referred to as “TAXOTERE”. The structures of otherpaclitaxel analogs are shown in FIGS. 3-23. These compounds have thebasic taxane skeleton as a common structure feature and have also beenshown to have the ability to arrest cells in the G2-M phases due tostabilization of microtubules. Thus, it is apparent from FIGS. 3-23 thata wide variety of substituents can decorate the taxane skeleton withoutadversely affecting biological activity. It is also apparent that zero,one or both of the cyclohexane rings of a paclitaxel analog can have adouble bond at the indicated positions. For clarity purposes, the basictaxane skeleton is shown below in Structural Formula (VI):

Double bonds have been omitted from the cyclohexane rings in the taxaneskeleton represented by Structural Formula (VI). The basic taxaneskeleton can include zero or one double bond in one or both cyclohexanerings, as indicated in FIGS. 3-23 and Structural Formulas (VII) and(VIII) below. A number of atoms have also been omitted from StructuralFormula (VI) to indicate sites in which structural variation commonlyoccurs among paclitaxel analogs. For example, substitution on the taxaneskeleton with simply an oxygen atom indicates that hydroxyl, acyl,alkoxy or another oxygen-bearing substituent is commonly found at thesite. These and other substitutions on the taxane skeleton can be madewithout losing the ability to enhance and stabilize microtubuleformation. Thus, the term “paclitaxel analog” is defined herein to meana compound which has the basic paclitaxel skeleton and which promotesmicrotubule formation. paclitaxel analogs may be formulated as ananoparticle colloidal composition to improve the infusion time and toeliminate the need to deliver the drug with Cremophor which causeshypersensitivity reactions in some patients. An example of a paclitaxelanalog formulated as a nanoparticle colloidal composition is ABI-007which is a nanoparticle colloidal composition of protein-stabilizedpaclitaxel that is reconstituted in saline.

Typically, the paclitaxel analogs used herein are represented byStructural Formula (VII) or (VIII):

R₁₀ is a lower alkyl group, a substituted lower alkyl group, a phenylgroup, a substituted phenyl group, —SR₁₉, —NHR₁₉ or —OR₁₉.

R₁₁ is a lower alkyl group, a substituted lower alkyl group, an arylgroup or a substituted aryl group.

R₁₂ is —H, —OH, lower alkyl, substituted lower alkyl, lower alkoxy,substituted lower alkoxy, —O—C(O)-(lower alkyl), —O—C(O)-(substitutedlower alkyl), —O—CH₂—O-(lower alkyl) —S—CH₂—O-(lower alkyl).

R₁₃ is —H, —CH₃, or, taken together with R₁₄, —CH₂—.

R₁₄ is —H, —OH, lower alkoxy, —O—C(O)-(lower alkyl), substituted loweralkoxy, —O—C(O)-(substituted lower alkyl), —O—CH₂—O—P(O)(OH)₂,—O—CH₂—O-(lower alkyl), —O—CH₂—S-(lower alkyl) or, taken together withR₂₀, a double bond.

R₁₅ —H, lower acyl, lower alkyl, substituted lower alkyl, alkoxymethyl,alkthiomethyl, —OC(O)—O(lower alkyl), —OC(O)—O(substituted lower alkyl),—OC(O)—NH(lower alkyl) or —OC(O)—NH(substituted lower alkyl).

R₁₆ is phenyl or substituted phenyl.

R₁₇ is —H, lower acyl, substituted lower acyl, lower alkyl, substituted,lower alkyl, (lower alkoxy)methyl or (lower alkyl)thiomethyl.

R₁₈ —H, —CH₃ or, taken together with R₁₇ and the carbon atoms to whichR₁₇ and R₁₈ are bonded, a five or six membered a non-aromaticheterocyclic ring.

R₁₉ is a lower alkyl group, a substituted lower alkyl group, a phenylgroup, a substituted phenyl group.

R₂₀ is —H or a halogen.

R₂₁ is —H, lower alkyl, substituted lower alkyl, lower acyl orsubstituted lower acyl.

Preferably, the variables in Structural Formulas (VII) and (VIII) aredefined as follows: R₁₀ is phenyl, tert-butoxy, —S—CH₂—CH—(CH₃)₂,—S—CH(CH₃)₃, —S—(CH₂)₃CH₃, —O—CH(CH₃)₃, —NH—CH(CH₃)₃, —CH═C(CH₃)₂ orpara-chlorophenyl; R₁₁ is phenyl, (CH₃)₂CHCH₂—, -2-furanyl, cyclopropylor para-toluyl; R₁₂ is —H, —OH, CH₃CO— or —(CH₂)₂—N-morpholino; R₁₃ ismethyl, or, R₁₃ and R₁₄, taken together, are —CH₂—;

R₁₄ is —H, —CH₂SCH₃ or —CH₂—O—P(O)(OH)₂; R₁₅ is CH₃CO—;

R₁₆ is phenyl; R₁₇ —H, or, R₁₇ and R₁₈, taken together, are —O—CO—O—;

R₁₈ is —H; R₂₀ is —H or —F; and R₂, is —H, —C(O)—CHBr—(CH₂)₁₃—CH₃ or—C(O)—(CH₂)₁₄—CH₃; —C(O)—CH₂—CH(OH)—COOH,—C(O)—CH₂—O—C(O)—CH₂CH(NH₂)—CONH₂, —C(O)—CH₂—O—CH₂CH₂OCH₃ or—C(O)—O—C(O)—CH₂CH₃.

A paclitaxel analog can also be bonded to or be pendent from apharmaceutically acceptable polymer, such as a polyacrylamide. Oneexample of a polymer of this type is shown in FIG. 24. The term“paclitaxel analog”, as it is used herein, includes such polymers.

The bis(thio-hydrazide amide) disalts disclosed herein can be preparedby a method of the invention. The method of preparing the disclosedbis(thio-hydrazide amide) disalts includes the steps of combining aneutral bis(thio-hydrazide amide), an organic solvent and a base to forma bis(thio-hydrazide amide) solution; and combining the solution and anorganic antisolvent, thereby precipitating a disalt of thebis(thio-hydrazide amide) (e.g., compounds represented by StructuralFormulas (I)-(V)). The neutral forms of the disclosed bis(thio-hydrazideamide) disalts can be prepared according to methods described in U.S.Publication Nos. 2003/0045518 and 2003/0119914, both entitled SYNTHESISOF TAXOL ENHANCERS and also according to methods described in U.S.Publication No. 2004/0225016 A1, entitled TREATMENT FOR CANCERS. Theentire teachings of these publications are incorporated herein byreference.

Typically, at least about two molar equivalents of the base are employedfor each molar equivalent of neutral bis(thio-hydrazide amide); moretypically, from about 2 to about 5 equivalents, or preferably from about2.0 to about 2.5 equivalents.

Suitable bases can be strong enough to react with a bis(thio-hydrazideamide) to produced a disalt. In various embodiments, the base can be anamine (e.g., triethylamine, diphenylamine, butylamine, or the like); anammonium hydroxide (e.g., tetramethyammonium hydroxide,tetrabutylammonium hydroxide, or the like); an alkali metal hydroxide(lithium hydroxide, sodium hydroxide, potassium hydroxide, or the like)an alkali metal C1-C6 alkoxide, or an alkali metal amide (e.g., sodiumamide, lithium diisopropyl amide, or the like). In some embodiments, thebase is sodium hydroxide, potassium hydroxide, sodium C1-C6 alkoxide,potassium C1-C6 alkoxide, sodium amide, or potassium amide, orpreferably, sodium hydroxide, sodium methoxide, or sodium ethoxide.

In various embodiments, the base can be an alkali metal hydride (e.g.,sodium hydride, potassium hydride, or the like), a divalent metal base(e.g., magnesium oxide) a C1-C6 alkyl alkali metal (e.g., butyllithium),or an aryl alkali metal (e.g., phenyllithium). More typically, the baseis lithium hydride, sodium hydride, potassium hydride, butyllithium,butylsodium, butylpotassium, phenyllithium, phenylsodium, orphenylpotassium.

As used herein, an alkali metal includes lithium, sodium, potassium,cesium and rubidium.

The organic solvent can be any organic solvent which is stable when thebase is added to a mixture of the bis(thio-hydrazide amide) and theorganic solvent. Typically, the organic solvent is polar enough todissolve the bis(thio-hydrazide amide) salt formed by the method to forma solution. In various embodiments, the organic solvent iswater-miscible. The organic solvent can generally be selected from aC1-C4 aliphatic alcohol (e.g., methanol, ethanol, 1-propanol,2-propanol, or the like), a C1-C4 aliphatic ketone (e.g., acetone,methyl ethyl ketone, 2-butanone, or the like), a C2-C4 aliphatic ether(e.g., diethyl ether, dipropyl ether, diisopropyl ether, or the like), aC2-C4 cycloaliphatic ether (e.g., tetrahydrofuran, dioxane, or thelike), dimethyl formamide, dimethyl sulfoxide, N-methyl pyrrolidone, aglycol (e.g., ethylene glycol, propylene glycol, tetramethylene glycol,or the like), an alkyl glycol ether (e.g., ethylene glycol dimethylether, or the like), and acetonitrile. More typically, the organicsolvent can be selected from methanol, ethanol, propanol (e.g.,1-propanol, 2-propanol), butanol (e.g., 1-butanol, tert-butyl alcohol,or the like), acetone, tetrahydrofuran, and methyl ethyl ketone.Preferably, the organic solvent can be selected from methanol, ethanol,acetone, and methyl ethyl ketone.

As used herein, the organic antisolvent is a solvent that when added tothe solution created by combining the base, the bis(thio-hydrazideamide) and the organic solvent, causes the bis(thiohydrazide amide)disalt to precipitate out of solution. Typically, the organicantisolvent can be selected from a C5-C10 alkane (e.g., pentane,petroleum ether, hexane, heptane, octane, isooctane, or the like),C5-C10 cycloalkane (e.g., cyclohexane, cyclopentane, or the like), aC3-C10 alkyl ester (e.g., ethyl acetate, propyl acetate, methylbutyrate, or the like, a C3-C10 alkyl ether (e.g., methyl ethyl ether,diethyl ether, methyl propyl ether, or the like), benzene, toluene, andxylene. More typically, the organic antisolvent can be selected fromdiethyl ether, dipropyl ether (e.g., propyl as 1-propyl or 2-propyl),methyl propyl ether, ethyl propyl ether, methyl tert-butyl ether, methylacetate, ethyl acetate, propyl acetate, pentane, hexane, cyclohexane,heptane, and petroleum ether. In some embodiments, the organicantisolvent can be a C5-C10 alkane or C5-C10 cycloalkane. In variouspreferred embodiments, the organic antisolvent can be heptane; or, theorganic antisolvent can be diethyl ether or ethyl acetate. In variouspreferred embodiments, the organic antisolvent can be methyl tert-butylether.

In various embodiments, the neutral bis(thio-hydrazide amide) can besubstantially insoluble in the organic solvent, thereby forming amixture, whereby combining the base with the mixture forms abis(thio-hydrazide amide) solution. Typically, the bis(thio-hydrazideamide) solution can be clear. Generally, between about 0.25 and about2.5 moles of the neutral bis(thio-hydrazide amide) are combined per eachliter of organic solvent, or typically between about 0.75 and about 1.5moles of the neutral bis(thio-hydrazide amide) are combined per eachliter of organic solvent. Preferably, about 1 mole of the neutralbis(thio-hydrazide amide) are combined per each liter of organicsolvent.

As used herein, a “bis(thio-hydrazide amide) solution,” when formed fromthe organic solvent, the neutral bis(thio-hydrazide amide), and thebase, can include one or more species such as the neutralbis(thio-hydrazide amide), the bis(thio-hydrazide amide) monosalt, thebis(thio-hydrazide amide) disalt, or the like.

In preferred embodiments, the organic solvent is ethanol. Preferably,the base is about 2 molar to about 5 molar aqueous sodium hydroxide, ormore preferably from about 2 to about 2.5 molar.

In preferred embodiments, the organic solvent is acetone. Preferably,the base is about 2 molar to about 5 molar ethanolic sodium ethoxide, ormore preferably from about 2 to about 2.5 molar.

The bis(thio-hydrazide amide) disalts prepared by the present inventionare the disalts disclosed herein, including those represented byStructural Formulas (I)-(V). The neutral bis(thio-hydrazide amides)employed in the disclosed method to prepare the disalts represented byStructural Formulas (I)-(II) can be represented by the followingStructural Formulas (I′)-(II′), where the variables have the same valuesand preferred values as in Structural Formulas (I)-(II), respectively:

Thus, as used herein, a neutral bis(thio-hydrazide amide) has at leasttwo hydrogens (e.g., the hydrogens bonded to the nitrogen atoms inStructural Formulas (I′) and (II′) which can react with the basesdescribed herein to form a disalt.

In Structural Formula (I), M⁺ is a pharmaceutically acceptablemonovalent cation. M²⁺ is a pharmaceutically acceptable divalent cationas described above.

In various preferred embodiments, the organic solvent can be acetone;the base can be ethanolic sodium ethoxide; the organic solvent can beethanol; the base can be aqueous sodium hydroxide; the antisolvent canbe heptane; the neutral bis(thio-hydrazide amide) can be:

and/or the neutral bis(thio-hydrazide amide) can be:

The present invention is illustrated by the following examples, whichare not intended to be limiting in any way.

EXEMPLIFICATION Example 1 Sodium;2-(N′-methyl-N′-thiobenzoyl-hydrazinocarbonyl)-1-(methyl-thiobenzoyl-hydrazono)-ethanolate:Compound (3)

To a stirred clear solution of N-Malonyl-bis(N′-thiobenzoyl-N′-methylhydrazide) (2 g) in THF (25 mL) was added sodium amide (0.2 g). Theresultant mixture was stirred at room temperature for 4 hours. Theprecipitated product was collected by filtration and washed with THF andether. The resulting solids were dried in vacuo (0.1 mmHg) for 12 hours.A light yellow solid weighing 1.53 g was obtained.

¹H-NMR (DMSO-d₆) δ (ppm): 2.2-2.7 (m, 2H), 3.1-3.7 (m, 6H), 7.1-7.5 (m,10 H). Elem. Anal. Calcd. For C₁₉H₁₉N₄NaO₂S₂·0.55H₂O: C 52.75%, H 4.66%,N12.98%; found: C 52.42%, H 4.41%, N 13.07%.

Example 2 Disodium:2-(N′-methyl-N′-thiobenzoyl-hydrazinocarbonyl)-1-(methyl-thiobenzoyl-hydrazono)-ethanolate:Compound (1)

Method A: To a stirred clear solution of NaOH (0.358 g, 8.95 mmol) inH₂O (10 mL) was added N-Malonyl-bis(N′-thiobenzoyl-N′-methyl hydrazide)(1.79 g, 4.48 mmol) at room temperature. The resultant solution wasfiltered, then the filtrate was cooled with a dry-ice/^(i)PrOH bathfollowed by freeze-drying to obtain Compound 1 as a solid.

¹H-NMR (DMSO-d₆) δ (ppm): 2.15 (s, 2H), 3.45 (s, 6H), 7.0-7.2 (m, 6 H),7.2-7.4 (m, 4H). Elem. Anal. Calcd for C₁₉H₁₈N₄Na₂O₂S₂.0.7H₂O (457.22):C 49.91; H 4.29; N12.26; found: C 49.95; H 4.17; N12.05.

Method B: To a stirred solutionof-N-Malonyl-bis(N′-thiobenzoyl-N′-methyl hydrazide) (0.92 g) in THF (15mL) was added a solution of NaOH (0.184 g) in MeOH (15 mL). After 15 minstirring at room temperature, the resultant solution was concentratedand precipitated with ether to yield 1 g of a light orange powder afterfiltration. The physical data for the precipitate was is in accordancewith that obtained from Method A.

Example 3 Lithium;2-(N′-methyl-N′-thiobenzoyl-hydrazinocarbonyl)-1-(methyl-thiobenzoyl-hydrazono)-ethanolate:Compound (4)

To a solution of N-Malonyl-bis(N′-thiobenzoyl-N′-methyl hydrazide) (8 g,20 mmol) in THF (90 ml) was added llithium diisopropylamide (LDA) (20mmol, 2M in heptane/THF/ethylbenzene) at 0° C., and the mixture wasstirred for 2 hours. The precipitate was collected, washed with EtOAc,and dried to give a mono lithium salt ofN-Malonyl-bis(N′-thiobenzoyl-N′-methyl hydrazide) (6.5 g).

¹H-NMR (DMSO-d₆) δ (ppm), 7.71-7.32 (m, 10H), 3.72-2.73(m, 9H).

Example 4 Dilithium;2-(N′-methyl-N′-thiobenzoyl-hydrazinocarbonyl)-1-(methyl-thiobenzoyl-hydrazono)-ethanolate:Compound (5)

Compound 5 was prepared according to the method described in Example 3except that 2 equivalents of LDA were used instead of one equivalent.

¹H-NMR (DMSO-d₆) δ (ppm): 7.61-7.12 (m, 10H), 3.82-2.29(m, 8H).

Example 5 Ethanolamine;2-(N′-methyl-N′-thiobenzoyl-hydrazinocarbonyl)-1-(methyl-thiobenzoyl-hydrazono)-ethanolate:Compound (6)

The monoethanolamine compound was made in a similar to that in Example 3by using one equivalent of ethanolamine instead of LDA.

¹H-NMR 4783 (DMSO-d₆) δ (ppm): 7.72-7.33 (m, 10H), 3.80-2.63(m, 13H).

Example 6 Diethanolamine;2-(N′-methyl-N′-thiobenzoyl-hydrazinocarbonyl)-1-(methyl-thiobenzoyl-hydrazono)-ethanolate:Compound (7)

The diethanolamine compound was made in a similar to that in Example 3by using two equivalents of ethanolamine instead of one equivalent ofLDA.

¹H-NMR 4783 (DMSO-d₆) δ (ppm): 7.69-7.31 (m, 10H), 3.78-2.64(m, 16H)

Example 7 Magnesium;2-(N′-methyl-N′-thiobenzoyl-hydrazinocarbonyl)-1-(methyl-thiobenzoyl-hydrazono)-ethanolate:Compound (8)

The pH of a suspension of MgO (440 mg, 1.1 mmol, 15 mL) in water wasadjusted to 2-3 to form a clear solution. To this solution was addeddilithium;2-(N′-methyl-N′-thiobenzoyl-hydrazinocarbonyl)-1-(methyl-thiobenzoyl-hydrazono)-ethanolate(1 mmol), and the mixture was stirred for 3 hours. The precipitate thatformed was collected and washed with water, and then dried to give themagnesium salt (2.1 g).

¹H-NMR (DMSO-d₆) δ (ppm): 7.68-7.32 (m, 10H), 3.98-2.49(m, 8H).

Example 8 Calcium;2-(N′-methyl-N′-thiobenzoyl-hydrazinocarbonyl)-1-(methyl-thiobenzoyl-hydrazono)-ethanolate:Compound (9)

The calcium compound was made in a similar to that in Example 7 by usingCa(OH)₂ instead of MgO.

¹H-NMR (DMSO-d₆) δ (ppm): 7.67-7.34 (m, 10H), 3.91-2.55(m, 8H).

Example 9 Monopotassium;2-(N′-methyl-N′-thiobenzoyl-hydrazinocarbonyl)-1-(methyl-thiobenzoyl-hydrazono)-ethanolate:Compound (10)

N-Malonyl-bis(N′-thiobenzoyl-N′-methyl hydrazide) (micronized), 5.006 g(12.5 mmol) was suspended in 70 mL of absolute ethanol. Into thissuspension was added with stirring 12.4 mL (1 equivalent) of a 1.008 Nsolution of potassium hydroxide in methanol. The resulting solution wasfiltered through a glass acrodisc (Gelman) and concentrated to about 5mL. Into this concentrated solution, 5 mL of ethyl acetate was added,followed by anhydrous ether until the resulting solution turned cloudy.This solution was left for crystallization overnight. A hardenedprecipitate was broken up with a spatula, filtered out, washed twicewith anhydrous ether and vacuum-dried at 50° C. to afford amono-potassium salt as pale-yellow solids (4.05 g, 70%).

¹H NMR (DMSO-d₆) (a mixture of tautomers): δ (ppm): 7.4-7.25, 7.22 (m,10H), 3.60, 3.45 and 3.19 (singlets, 6H), 2.78, 2.39 and 1.96 (singlets,3H) ). IR (KBr): 1686s, 1572, 1478 cm⁻¹. Anal. Calcd forC₁₉H₁₉K₂N₄O₂S₂+H₂O: C, 49.98; H, 4.64; K, 8.56; N, 12.27. Found: C,49.99; H, 4.51; K, 8.67; N, 11.91.

Example 10 Dipotassium;2-(N′-methyl-N′-thiobenzoyl-hydrazinocarbonyl)-1-(methyl-thiobenzoyl-hydrazono)-ethanolate:Compound (2)

Method A: N-Malonyl-bis(N′-thiobenzoyl-N′-methyl hydrazide) (micronized,5.006 g, 12.5 mmol) was treated with 24.95 mL (2 equivalent) of 1.002 Nsolution of potassium hydroxide in water (volumetric standard solution,Aldrich). All material except a few particles dissolved, and 6 mL ofMilli-Q water was added to afford a solution with some tiny particlesremaining. The solution was then filtered through a glass acrodisc(Gelman), the filter was rinsed with 2 mL of Milli-Q water, and thecombined solution was diluted to 50 mL with Milli-Q water andlyophilized to produce a title compound as yellow foams (6.17 g).

¹H NMR (DMSO-d₆): δ (ppm): 7.34-7.31 (m, 4H), 7.11-7.09 (m, 6H), 3.51(m, 6H), 2.11 (s, 2H). IR (KBr): 1561, cm⁻¹. Anal. Calcd forC₁₉H₁₈K₂N₄O₂S₂+H₂O; C, 46.13; H, 4.07; K, 15.81; N, 11.33. Found: C,46.08; H, 4.21; K, 16.05; N, 11.32.

Method B: Alternatively, N-Malonyl-bis(N′-thiobenzoyl-N′-methylhydrazide) (micronized, 5.006 g, 12.5 mmol) was dissolved in anhydrousTHF (62 mL). Into this solution, and 25.1 mL (2 equivalent) of 1.008Nsolution of potassium hydroxide in methanol (volumetric standardsolution, Aldrich) was added. From the resulting solution, solvent wasremoved under reduced pressure to leave about 5 mL of an oily residue,which was triturated with anhydrous ether until a pale yellow solid wasobtained. The solid was filtered out, washed twice with anhydrous etherand vacuum-dried at 50° C., to yield the dipotassium salt as a poweder(4.3 g, 73%). NMR data were identical with those obtained above inMethod A.

Example 11 Cholin;2-(N′-methyl-N′-thiobenzoyl-hydrazinocarbonyl)-1-(methyl-thiobenzoyl-hydrazono)-ethanolate:Compound (11)

Into a stirred solution of N-Malonyl-bis(N′-thiobenzoyl-N′-methylhydrazide) (2 g, 5 mmol) in 65 mL of anhydrous THF was added dropwise1.32 mL of a solution of choline hydroxide (45% solution in MeOH) in 10ml of anhydrous THF. At first, a clear solution was formed, followed byprecipitation of a fine solid. The solution was left for 1 hour for theprecipitation to be completed. The precipitate was filtered out, washedtwice with ether:THF (2:1, v/v), then once with anhydrous ether, andvacuum-dried at 50° C. to obtain the titled salt as a pale yellow powder(2.14 g, 85%).

¹H NMR (DMSO-d₆) (a mixture of tautomers): δ (ppm): 7.4-7.35, 7.24-7.19(m, 10H), 3.83-3.80 (m, 2H), 3.40-3.37 (m, 2H), 3.60, 3.45 and 3.19(singlets, 6H), 3.10 (m, 9H), 2.64, 2.32 and 2.06 (singlets, 3H) ). IR(KBr): 1686s, 1586s, 1482s cm⁻¹.

Example 12 Disodium2-(N′-methyl-N′-thio-1-methylcyclopropyl-hydrazinocarbonyl)-1-(methyl-thio-1-methylcyclopropyl-hydrazono)-ethanolate:Compound (12)

Into a stirred clear solution of NaOH (24.7 mg, 0.62 mmol) in H₂O (5 mL)was added N-Malonyl-bis(N′-thio-1-methylcyclopropyl-N′-methyl hydrazide)(110 mg, 0.31 mmol) at room temperature. The resultant solution wasfiltered, then the filtrate was cooled with a dry-ice/^(i)PrOH bathfollowed by freeze-drying to obtain Compound (12) as a solid.

¹H-NMR (DMSO-d₆) δ (ppm): 0.38 (t, 4H, J=5), 0.83 (t, 4H, J=5), 1.18 (s,6 H), 2.62 (s, 2H), 3.38 (s, 6H).

Example 13 Disodium;2-(N′-methyl-N′-thiocyclopropyl-hydrazinocarbonyl)-1-(methyl-thiocyclopropyl-hydrazono)-ethanolate:Compound (13)

The disodium compound was prepared by a similar method to that used forExample 12.

¹H-NMR (DMSO-d₆) δ (ppm): 0.45 (m, 4H), 0.85 (m, 4H), 2.61 (s, 2 H),2.70 (m, 1H), 3.41 (s, 6H).

Example 14 Dipotassium:2-(N′-methyl-N′-thio-1-methylcyclopropyl-hydrazinocarbonyl)-1-(methyl-thio-1-methylcyclopropyl-hydrazono)-ethanolate:Compound (14)

The dipotassium compound was prepared by a method similar to that usedfor Example 12 except that KOH was used instead of NaOH.

¹H-NMR (DMSO-d₆) δ (ppm): 0.39 (m, 4H), 0.82 (m, 4H), 1.18 (s, 6 H),2.60 (s, 2H), 3.38 (s, 6H).

Example 15 Bis (Thio-Hydrazide Amide) Disalts Have Significantly GreaterSolubility Than The Corresponding Monosalts And Neutral Forms

A compound was weighed and water was added to the compound such that theresulting concentration of the compound was about 100 mg/mL. The mixturewas then shaken well and sonicated (about 5-10 min at about 50° C.). Ifthe added amounts of the compound were totally solubilized, more amountsof the compound were added to the solution and the mixture was shakenwell and sonicated until a suspension was achieved. The suspension wasthen filtered through a 0.2 μm filter. The filtered solution was dilutedwith DMSO. Then the mixture was analyzed by HPLC to determine theconcentration of the compound.

The HPLC system consisted of the HP 1100 Model (Agilent, Wilmington,Del.) equipped with a model 1100 quaternary pump, a model 1100autosampler, a model 1100 Diode-Array Detector for UV detection at 280nm. HPLC analysis was performed using a gradient mobile phase consistingof acetonitrile-water containing 0.1% formic acid. Mobile phases weredegassed and filtered through a solvent filtration apparatus and pumpedat a constant rate of 1.0 mL/min. Separation was made on an XTerra MSC18 analytical column, 4.6 mm i.d.×150 mm, (Waters Corp., Milford,Mass., USA) fitted with a pre-column filter (XTerra MS C18, 3.9 mm×20mm). The column was maintained at 40° C. Data acquisition and instrumentsetting were controlled using HP Chemstation software (version 8.03).

Solubility data for each compound tested are shown below in Table 1.

TABLE 1 Solubility Data Samples Salts Compound Solubility in water(mg/mL) A — Neutral Form <0.1 of Compound 1 B 2Na⁺ 1 >1000 C Li⁺ 4 53.5D 2Li⁺ 5 626.0 (>500) E  Na⁺ 3 52.8 F  2K⁺ 2 >1000 G K⁺ 10 41.2 H Ca²⁺ 918.9 I  HEA⁺ 6 51.5 J  2HEA⁺ 7 231.1 (>200) K Mg²⁺ 8 43.5 L  Choline⁺ 11264.0 (>200) ^( M) 2Na⁺ 12 >1000 N 2Na⁺ 13 >1000 O 2K⁺ 14 >1000

As can be seen in Table 1, the bis(thio-hydrazide amide) disalts, forexample, Compounds 1, 2, 5, 7, 12, 13 and 14, have significantly greaterwater solubility than the corresponding neutral or monosalt compounds.In particular, Compounds 1 and 2 showed significantly greater watersolubility than the corresponding neutral form. Similarly, disaltscompounds, Compounds 1, 2, 5 and 7, showed much greater water solubilitythan the corresponding monosalt compounds, Compounds 3, 4, 6 and 10.

Example 16 Bis (Thio-Hydrazide Amide) Disalts Have Significantly GreaterBioavailability Than The Corresponding Monosalts And Neutral Forms

A) Procedure for Pharmacokinetics Study in Dog

Male beagle dogs were acclimated for at least one week prior to use,were fed laboratory chow and water ad libitum, and were housed intemperature and humidity controlled rooms. Compounds were prepared as asolution for intravenous injection or a capsule for oral administration.Three dogs were used for the study. Compounds were injectedintravenously via cephalic vein or by oral gavage. The dose was adjustedbased on the animal's body weight. Blood samples were collected atintervals of 5, 10 and 30 min and 1, 2, 4, 6, 8 and 24 hr (example)after administration of the compound and plasma samples were preparedafter centrifugation (5000 rpm, 8 min) of whole blood samples. Compoundin plasma was measured by a liquid chromatography with tandem massspectrophotometer (LC/MS/MS) after 50 μl of these samples were extractedby protein precipitation with acetonitrile. The compound concentrationwas determined by the standard curve (concentration vs. peak area) madewith the same extraction procedure of controlled plasma. The area undercurve (AUC) was calculated using the modified trapezoidal method. Theportion of AUC from the last measurable plasma concentration to infinitywas estimated by C/k, where k was expressed by the least squareregression of the log-linear concentration time points and C representsthe last measurable plasma concentration. Bioavailability was calculatedby AUCpo/AUCiv.

B) Bioanalytical Method of Compounds (1) and (3) in Plasma by LC/MS/MS

Compounds (1) and (3) and its internal standard were extracted fromheparinized plasma by protein precipitation with acetonitrile.Chromatography was achieved on an XTerra column (Waters, particle size:5 μm; 100 mm×3.0 mm i.d.) using a mixture of acetonitrile, water andformic acid. Analysis was performed on a Sciex API 365 tandem massspectrometer with turbo ion spray interface. Negative ions were measuredusing the multiple-reaction-monitoring (MRM) mode with m/z 399.0→165.1.The run time was 9 min per sample, and Compounds (1) and (3) werequantitated by a peak area ratio using 1/X²-weighted least-square linearregression plot.

Bioavailability data for each compound tested are shown below in Table2.

TABLE 2 Bioavailability Data Bioavailability Samples Salts Compounds (%)1 — Neutral Form of 4.8 Compound (1) 2 2Na⁺ 1 80 3 Na⁺ 3 35

As can be seen in Table 2, the disalt compound, Compound (1), showedmuch greater bioavailability than the corresponding neutral compound ormonosalt compound, Compound (3).

Example 17 Process Method for Preparing Disodium Salts

I. General Method

About 50 mmol of the neutral form of a compound of the invention issuspended in 50 mL of acetone. About 2.04 equivalents of NaOH isdissolved in ethanol to form 38 mL of a 21% solution. The NaOH solutionis added to the suspension of the compound while maintaining thetemperature at about 0° C. The mixture is stirred for about 20 min. togive a clear solution, then about 1.2 mL of water is add. The solutionis allowed to come to room temperature and about 400 mL of heptane isadded. The solution is allowed to stir about 12 hours and the resultingprecipitate is collected by filtration.

The precipitate is added to about 60 mL of acetone and stirred for 2hours at room temperature, then collected by filtration and dried undervacuum for 1 hour at about 50° C. to yield the pure disodium salt of thecompound.

II. Preparation of Disodium;2-(N′-methyl-N′-thiobenzoyl-hydrazinocarbonyl)-1-(methyl-thiobenzoyl-hydrazono)-ethanolate:Compound (1)

Compound (1) was prepared by the above general method. The analyticaldata was as follows:

¹H NMR(DMSO-d₆) (ppm) 7.38-7.02 (m, 10H), 3.51 (s, 6H), 2.13 (s, 2H)

Elemental Analysis: Calculated for C₁₉H₂₀N₄Na₂O₂S₂: C 49.34, H 4.36, N12.11, S 13.87; found C 49.28, H 4.18, N 11.91, S 13.63

Examples 18-20 Preparation of Disodium Bis(thio-hydrazide amide)Solution

A sample of a bis(thio-hydrazide amide) (Compound 1, 15 grams) wascombined with 40 mL of absolute ethanol to form a mixture as a slurry.Aqueous sodium hydroxide (3.0 grams NaOH in 3.0 mL H20) was added to themixture with stirring at room temperature, and the mixture was cooled tonot exceed 35 degrees C. The aqueous sodium hydroxide addition vesselwas rinsed with 1 mL of water and 5 mL of ethanol, and the rinses wereadded to the mixture. After addition, the mixture was stirred for 110minutes. The resulting yellow disodium bis(thio-hydrazide amide)solution was separated into three equal portions for the followingexamples.

Example 18 63% Yield of Bis(thio-hydrazide amide) Disodium Salt

A one-third portion of the above yellow disodium bis(thio-hydrazideamide) solution was combined with 17 mL of methyl tert-butyl ether andstirred for 60 minutes (precipitation occurred in less than 30 minutes).The resulting slurry was filtered, washed with 10 mL of a 1:1 mixture ofethyl acetate:methyl tert-butyl ether, followed by 5 mL of ethylacetate. Residual solvent was removed by vacuum to give 3.51 grams (63%)of the disodium salt of Compound (1) as a pale yellow solid. A yellowcontaminant was visible.

Example 19 87% Yield of Pure Bis(thio-hydrazide amide) Disodium Salt

A one-third portion of the above yellow disodium bis(thio-hydrazideamide) solution was combined with 17 mL of methyl tert-butyl ether andstirred for 60 minutes (precipitation occurred in less than 30 minutes).An additional 17 mL of methyl tert-butyl ether was added to theresulting thick slurry, and was stirred for an additional 14 hours. Theresulting slurry was filtered, washed with 10 mL of a 1:1 mixture ofethyl acetate:methyl tert-butyl ether, followed by 10 mL of ethylacetate. Residual solvent was removed by vacuum to give 4.84 grams (87%)of the disodium salt of Compound (1) as a pale yellow solid. No yellowcontaminant was visible.

Example 20 96% Yield of Pure Bis(thio-hydrazide amide) Disodium Salt

A one-third portion of the above yellow disodium bis(thio-hydrazideamide) solution was combined with 17 mL of methyl tert-butyl ether andstirred for 60 minutes (precipitation occurred in less than 30 minutes).An additional 34 mL of methyl tert-butyl ether was added to theresulting thick slurry, and was stirred for an additional 14 hours. Theresulting slurry was filtered, washed with 10 mL of a 1:1 mixture ofethyl acetate:methyl tert-butyl ether, followed by 10 mL of ethylacetate. Residual solvent was removed by vacuum to give 5.35 grams (96%)of the disodium salt of Compound (1) as a pale yellow solid. No yellowcontaminant was visible.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1. A compound represented by the following structural formula:

wherein: Y is a covalent bond or a substituted or unsubstituted straightchained hydrocarbyl group; R₁-R₄ are independently —H, an aliphaticgroup, a substituted aliphatic group, an aryl group or a substitutedaryl group, or R₁ and R₃ taken together with the carbon and nitrogenatoms to which they are bonded, and/or R₂ and R₄ taken together with thecarbon and nitrogen atoms to which they are bonded, form a non-aromaticheterocyclic ring optionally fused to an aromatic ring; Z is —O or —S;and M⁺ is a pharmaceutically acceptable monovalent cation and M²⁺ is apharmaceutically acceptable divalent cation.
 2. The compound of claim 1,wherein the compound is represented by the following structural formula:

wherein M⁺ is Na⁺ or K³⁰ .
 3. The compound of claim 2, wherein Z is O;R₁ and R₂ are the same; and R₃ and R₄ are the same.
 4. The compound ofclaim 3, wherein: Y is a covalent bond, —C(R₅R₆)—, —(CH₂CH₂)—,trans-(CH═CH)—, cis-(CH═CH)— or —(CC)— group; and R₅ and R₆ are eachindependently —H, an aliphatic or substituted aliphatic group, or R₅ is—H and R₆ is a substituted or unsubstituted aryl group, or R₅ and R₆,taken together, are a C2-C6 substituted or unsubstituted alkylene group.5. The compound of claim 4, wherein: Y is —C(R₅R₆)—; R₁ and R₂ are eacha substituted or unsubstituted aryl group; and R₃ and R₄ are each asubstituted or unsubstituted aliphatic group.
 6. The compound of claim5, wherein R₅ is —H and R₆ is —H, an aliphatic or substituted aliphaticgroup; R₃ and R₄ are each an alkyl group; and R₆ is —H or methyl.
 7. Thecompound of claim 6, wherein R₁ and R₂ are each a substituted orunsubstituted phenyl group and R₃ and R₄ are each methyl or ethyl. 8.The compound of claim 4, wherein Y is —CR₅R₆—; R₁ and R₂ are both asubstituted or unsubstituted aliphatic group; R₅ is —H; and R₆ is —H oran optionally substituted aliphatic group.
 9. The compound of claim 8,wherein R₁ and R₂ are both a C3-C8 cycloalkyl group optionallysubstituted with at least one alkyl group; R₃ and R₄ are both an alkylgroup; and R₆ is —H or methyl.
 10. The compound of claim 9, wherein R₁and R₂ are both cyclopropyl or 1-methylcyclopropyl.
 11. The compound ofclaim 1, wherein the compound is represented by the following structuralformula:

wherein: R₁ and R₂ are both phenyl; R₃ and R₄ are both methyl; R₅ and R₆are both —H; R₁ and R₂ are both phenyl; R₃ and R₄ are both ethyl; R₅ andR₆ are both —H; R₁ and R₂ are both 4-cyanophenyl; R₃ and R₄ are bothmethyl; R₅ is methyl; R₆ is —H; R₁ and R₂ are both 4-methoxyphenyl; R₃and R₄ are both methyl; R₅ and R₆ are both —H; R₁ and R₂ are bothphenyl; R₃ and R₄ are both methyl; R₅ is methyl; R₆ is —H; R₁ and R₂ areboth phenyl; R₃ and R₄ are both ethyl; R₅ is methyl; R₆ is —H; R₁ and R₂are both 4-cyanophenyl; R₃ and R₄ are both methyl; R₅ and R₆ are both—H; R₁ and R₂ are both 2,5-dimethoxyphenyl; R₃ and R₄ are both methyl;R₅ and R₆ are both —H; R₁ and R₂ are both 2,5-dimethoxyphenyl; R₃ and R₄are both methyl; R₅ is methyl; R₆ is —H; R₁ and R₂ are both3-cyanophenyl; R₃ and R₄ are both methyl; R₅ and R₆ are both —H; R₁ andR₂ are both 3-fluorophenyl; R₃ and R₄ are both methyl; R₅ and R₆ areboth —H; R₁ and R₂ are both 4-chlorophenyl; R₃ R₄ are both methyl; R₅ ismethyl; R₆ is —H; R₁ and R₂ are both 2-dimethoxyphenyl; R₃and R₄ areboth methyl; R₅ and R₆ are both —H; R₁ and R₂ are both 3-methoxyphenyl;R₃ and R₄ are both methyl; R₅ and R₆ are both —H; R₁ and R₂ are both2,3-dimethoxyphenyl; R₃ and R₄ are both methyl; R₅ and R₆ are both —H;R₁ and R₂ are both 2,3-dimethoxyphenyl; R₃ and R₄ are both methyl; R₅ ismethyl; R₆ is —H; R₁ and R₂ are both 2,5-difluorophenyl; R₃ and R₄ areboth methyl; R₅ and R₆ are both —H; R₁ and R₂ are both2,5-difluorophenyl; R₃ and R₄ are both methyl; R₅ is methyl; R₆ is —H;R₁ and R₂ are both 2,5-dichlorophenyl; R₃ and R₄ are both methyl; R₅ andR₆ are both —H; R₁ and R₂ are both 2,5-dimethylphenyl; R₃ and R₄ areboth methyl; R₅ and R₆ are both —H; R₁ and R₂ are both2,5-dimethoxyphenyl; R₃ and R₄ are both methyl; R₅ and R₆ are both —H;R₁ and R₂ are both phenyl; R₃ and R₄ are both methyl; R₅ and R₆ are both—H; R₁ and R₂ are both 2,5-dimethoxyphenyl; R₃ and R₄ are both methyl;R₅ is methyl; R₆ is —H; R₁ and R₂ are both cyclopropyl; R₃ and R₄ areboth methyl; R₅ and R₆ are both —H; R₁ and R₂ are both cyclopropyl; R₃and R₄ are both ethyl; R₅and R₆ are both —H; R₁ and R₂ are bothcyclopropyl; R₃ and R₄ are both methyl; R₅ is methyl; R₆ is —H; R₁ andR₂ are both 1-methylcyclopropyl; R₃ and R₄ are both methyl; R₅ and R₆are both —H; R₁ and R₂ are both 1-methylcyclopropyl; R₃ and R₄ are bothmethyl; R₅ is methyl and R₆ is —H; R₁ and R₂ are both1-methylcyclopropyl; R₃ and R₄ are both methyl; R₅ is ethyl and R₆ is—H; R₁ and R₂ are both 1-methylcyclopropyl; R₃ and R₄ are both methyl;R₅ is n-propyl and R₆ is —H; R₁ and R₂ are both 1-methylcyclopropyl; R₃and R₄ are both methyl; R₅ and R₆ are both methyl; R₁ and R₂ are both1-methylcyclopropyl; R₃ and R₄ are both ethyl; R₅ and R₆ are both —H; R₁and R₂ are both 1-methylcyclopropyl; R₃ is methyl, and R₄ is ethyl; R₅and R₆ are both —H; R₁ and R₂ are both 2-methylcyclopropyl; R₃ and R₄are both methyl; R₅ and R₆ are both —H; R₁ and R₂ are both2-phenylcyclopropyl; R₃ and R₄ are both methyl; R₅ and R₆ are both —H;R₁ and R₂ are both 1-phenylcyclopropyl; R₃ and R₄ are both methyl; R₅and R₆ are both —H; R₁ and R₂ are both cyclobutyl; R₃ and R₄ are bothmethyl; R₅ and R₆ are both —H; R₁ and R₂ are both cyclopentyl; R₃ and R₄are both methyl; R₅ and R₆ are both —H; R₁ and R₂ are both cyclohexyl;R₃ and R₄ are both methyl; R₅ and R₆ are both —H; R₁ and R₂ are bothcyclohexyl; R₃ and R₄ are both phenyl; R₅ and R₆ are both —H; R₁ and R₂are both methyl; R₃ and R₄ are both methyl; R₅and R₆ are both —H; R₁ andR₂ are both methyl; R₃ and R₄ are both t-butyl; R₅ and R₆ are both —H;R₁ and R₂ are both methyl; R₃ and R₄ are both phenyl; R₅ and R₆ are both—H; R₁ and R₂ are both t-butyl; R₃ and R₄ are both methyl; R₅ and R₆ areboth —H; R₁ and R₂ are ethyl; R₃ and R₄ are both methyl; R₅ and R₆ areboth —H; or R₁ and R₂ are both n-propyl; R₃ and R₄ are both methyl; R₅and R₆ are both —H.
 12. The compound of claim 11, wherein the compoundis represented by the following structural formula:


13. A pharmaceutical composition comprising a pharmaceuticallyacceptable carrier or diluent and a compound represented by thefollowing structural formula:

wherein: Y is a covalent bond or a substituted or unsubstituted straightchained hydrocarbyl group; R₁-R₄ are independently —H, an aliphaticgroup, a substituted aliphatic group, an aryl group or a substitutedaryl group, or R₁ and R₃ taken together with the carbon and nitrogenatoms to which they are bonded, and/or R₂ and R₄ taken together with thecarbon and nitrogen atoms to which they are bonded, form a non-aromaticheterocyclic ring optionally fused to an aromatic ring; Z is —O or —S;and M⁺ is a pharmaceutically acceptable monovalent cation and M²⁺ is apharmaceutically acceptable divalent cation.
 14. The pharmaceuticalcomposition of claim 13, wherein the compound is represented by thefollowing structural formula:

wherein M⁺ is Na⁺ or K⁺.
 15. The pharmaceutical composition of claim 14,wherein Z is O; R₁ and R₂ are the same; and R₃ and R₄ are the same. 16.The pharmaceutical composition of claim 15, wherein: Y is a covalentbond, —C(R₅R₆)—, —(CH₂CH₂)—, trans-(CH═CH)—, cis-(CH═CH)— or —(CC)—group; and R₅ and R₆ are each independently —H, an aliphatic orsubstituted aliphatic group, or R₅ is —H and R₆ is a substituted orunsubstituted aryl group, or, R₅ and R₆, taken together, are a C2-C6substituted or unsubstituted alkylene group.
 17. The pharmaceuticalcomposition of claim 16, wherein: Y is —C(R₅R₆)—; R₁ and R₂ are each asubstituted or unsubstituted aryl group; and R₃ and R₄ are each asubstituted or unsubstituted aliphatic group.
 18. The pharmaceuticalcomposition of claim 17, wherein R₅ is —H and R₆ is —H, an aliphatic orsubstituted aliphatic group; R₃ and R₄ are each an alkyl group; and R₆is —H or methyl.
 19. The pharmaceutical composition of claim 18, whereinR₁ and R₂ are each a substituted or unsubstituted phenyl group and R₃and R₄ are each methyl or ethyl.
 20. The pharmaceutical composition ofclaim 19, wherein Y is —CR₅R₆—; R₁ R₂ are both a substituted orunsubstituted aliphatic group; R₅ is —H; and R₆ is —H or an optionallysubstituted aliphatic group.
 21. The pharmaceutical composition of claim20, wherein R₁ and R₂ are both a C3-C8 cycloalkyl group optionallysubstituted with at least one alkyl group; R₃ and R₄ are both asubstituted an alkyl group; and R₆ is —H or methyl.
 22. Thepharmaceutical composition of claim 13, wherein the compound isrepresented by the following structural formula:

wherein: R₁ and R₂ are both phenyl; R₃ and R₄ are both methyl; R₅ and R₆are both —H; R₁ and R₂ are both phenyl; R₃ and R₄ are both ethyl; R₅ andR₆ are both —H; R₁ and R₂ are both 4-cyanophenyl; R₃ and R₄ are bothmethyl; R₅ is methyl; R₆ is —H; R₁ and R₂ are both 4-methoxyphenyl; R₃and R₄ are both methyl; R₅ and R₆ are both —H; R₁ and R₂ are bothphenyl; R₃ and R₄ are both methyl; R₅ is methyl; R₆ is —H; R₁ and R₂ areboth phenyl; R₃ and R₄ are both ethyl; R₅ is methyl; R₆ is —H; R₁ and R₂are both 4-cyanophenyl; R₃ and R₄ are both methyl; R₅ and R₆ are both—H; R₁ and R₂ are both 2,5-dimethoxyphenyl; R₃ and R₄ are both methyl;R₅ and R₆ are both —H; R₁ and R₂ are both 2,5-dimethoxyphenyl; R₃ and R₄are both methyl; R₅ is methyl; R₆ is —H; R₁ and R₂ are both3-cyanophenyl; R₃ and R₄ are both methyl; R₅ and R₆ are both —H; R₁ andR₂ are both 3-fluorophenyl; R₃ and R₄ are both methyl; R₅ and R₆ areboth —H; R₁ and R₂ are both 4-chlorophenyl; R₃ and R₄ are both methyl;R₅ is methyl; R₆ is —H; R₁ and R₂ are both 2-dimethoxyphenyl; R₃ and R₄are both methyl; R₅ and R₆ are both —H; R₁ and R₂ are both3-methoxyphenyl; R₃ and R₄ are both methyl; R₅ and R₆ are both —H; R₁and R₂ are both 2,3-dimethoxyphenyl; R₃ and R₄ are both methyl; R₅ andR₆ are both —H; R₁ and R₂ are both 2,3-dimethoxyphenyl; R₃ and R₄ areboth methyl; R₅ is methyl; R₆ is —H; R₁ and R₂ are both2,5-difluorophenyl; R₃ and R₄ are both methyl; R₅ and R₆ are both —H; R₁and R₂ are both 2,5-difluorophenyl; R₃ and R₄ are both methyl; R₅ ismethyl; R₆ is —H; R₁ and R₂ are both 2,5-dichiorophenyl; R₃ and R₄ areboth methyl; R₅ and R₆ are both —H; R₁ and R₂ are both2,5-dimethylphenyl; R₃ and R₄ are both methyl; R₅ and R₆ are both —H; R₁and R₂ are both 2,5-dimethoxyphenyl; R₃ and R₄ are both methyl; R₅ andR₆ are both —H; R₁ and R₂ are both phenyl; R₃ and R₄ are both methyl; R₅and R₆ are both —H; R₁ and R₂ are both 2,5-dimethoxyphenyl; R₃ and R₄are both methyl; R₅ is methyl; R₆ is —H; R₁ and R₂ are both cyclopropyl;R₃ and R₄ are both methyl; R₅ and R₆ are both —H; R₁ and R₂ are bothcyclopropyl; R₃ and R₄ are both ethyl; R₅ and R₆ are both —H; R₁ and R₂are both cyclopropyl; R₃ and R₄ are both methyl; R₅ is methyl; R₆ is —H;R₁ and R₂ are both 1-methylcyclopropyl; R₃ and R₄ are both methyl; R₅and R₆ are both —H; R₁ and R₂ are both 1-methylcyclopropyl; R₃ and R₄are both methyl; R₅ is methyl and R₆ is —H; R₁ and R₂ are both1-methylcyclopropyl; R₃ and R₄ are both methyl; R₅ is ethyl and R₆ is—H; R₁ and R₂ are both 1-methylcyclopropyl; R₃ and R₄ are both methyl;R₅ is n-propyl and R₆ is —H; R₁ and R₂ are both 1-methylcyclopropyl; R₃and R₄ are both methyl; R₅ and R₆ are both methyl; R₁ and R₂ are both1-methylcyclopropyl; R₃ and R₄ are both ethyl; R₅ and R₆ are both —H; R₁and R₂ are both 1-methylcyclopropyl; R₃ is methyl, and R₄ is ethyl; R₅and R₆ are both —H; R₁ and R₂ are both 2-methylcyclopropyl; R₃ and R₄are both methyl; R₅ and R₆ are both —H; R₁ and R₂ are both2-phenylcyclopropyl; R₃ and R₄ are both methyl; R₅ and R₆ are both —H;R₁ and R₂ are both 1-phenylcyclopropyl; R₃ and R₄ are both methyl; R₅and R₆ are both —H; R₁ and R₂ are both cyclobutyl; R₃ and R₄ are bothmethyl; R₅ and R₆ are both —H; R₁ and R₂ are both cyclopentyl; R₃ and R₄are both methyl; R₅ and R₆ are both —H; R₁ and R₂ are both cyclohexyl;R₃ and R₄ are both methyl; R₅ and R₆ are both —H; R₁ and R₂ are bothcyclohexyl; R₃ and R₄ are both phenyl; R₅ and R₆ are both —H; R₁ and R₂are both methyl; R₃ and R₄ are both methyl; R₅ and R₆ are both —H; R₁and R₂ are both methyl; R₃ and R₄ are both t-butyl; R₅ and R₆ are both—H; R₁ and R₂ are both methyl; R₃ and R₄ are both phenyl; R₅ and R₆ areboth —H; R₁ and R₂ are both t-butyl; R₃ and R₄ are both methyl; R₅ andR₆ are both —H; R₁ and R₂ are ethyl; R₃ and R₄ are both methyl; R₅ andR₆ are both —H; or R₁ and R₂ are both n-propyl; R₃ and R₄ are bothmethyl; R₅ and R₆ are both —H.
 23. The pharmaceutical composition ofclaim 22, wherein the compound is represented by the followingstructural formula:


24. A method of treating a subject with melanoma, said method comprisingadministering to the subject an effective amount of a compoundrepresented by the following structural formula:

wherein: Y is a covalent bond or a substituted or unsubstituted straightchained hydrocarbyl group; R₁-R₄ are independently —H, an aliphaticgroup, a substituted aliphatic group, an aryl group or a substitutedaryl group, or R₁ and R₃ taken together with the carbon and nitrogenatoms to which they are bonded, and/or R₂ and R₄ taken together with thecarbon and nitrogen atoms to which they are bonded, form a non-aromaticheterocyclic ring optionally fused to an aromatic ring; Z is —O or —S;and M⁺ is a pharmaceutically acceptable monovalent cation and M²⁺ is apharmaceutically acceptable divalent cation.
 25. The method of claim 24,wherein the melanoma is multi-drug resistant.
 26. The method of claim24, wherein the compound is co-administered with an anti-cancer agentthat stabilizes microtubules.
 27. The method of claim 24, wherein thecompound is co-administered with paclitaxel or an analog of paclitaxel.28. The method of claim 24, wherein: Z is O; R₁ and R₂ are the same; R₃and R₄ are the same; Y is a covalent bond, —C(R₅R₆)—, —(CH₂CH₂)—,trans-(CH═CH)—, cis-(CH═CH)— or —(CC)— group; and R₅ and R₆ are eachindependently —H, an aliphatic or substituted aliphatic group, or R₅ is—H and R₆ is a substituted or unsubstituted aryl group, or R₅ and R₆,taken together, are a C2-C6 substituted or unsubstituted alkylene group.29. The method of claim 28, wherein the compound represented by thefollowing structural formula:

wherein: R₁ and R₂ are both phenyl; R₃ and R₄ are both methyl; R₅ and R₆are both —H; R₁ and R₂ are both phenyl; R₃ and R₄ are both ethyl; R₅ andR₆ are both —H; R₁ and R₂ are both 4-cyanophenyl; R₃ and R₄ are bothmethyl; R₅ is methyl; R₆ is —H; R₁ and R₂ are both 4-methoxyphenyl; R₃and R₄ are both methyl; R₅ and R₆ are both —H; R₁ and R₂ are bothphenyl; R₃ and R₄ are both methyl; R₅ is methyl; R₆ is —H; R₁ and R₂ areboth phenyl; R₃ and R₄ are both ethyl; R₅ is methyl; R₆ is —H; R₁ and R₂are both 4-cyanophenyl; R₃ and R₄ are both methyl; R₅ and R₆ are both—H; R₁ and R₂ are both 2,5-dimethoxyphenyl; R₃ and R₄ are both methyl;R₅ and R₆ are both —H; R₁ and R₂ are both 2,5-dimethoxyphenyl; R₃ and R₄are both methyl; R₅ is methyl; R₆ is —H; R₁ and R₂ are both3-cyanophenyl; R₃ and R₄ are both methyl; R₅ and R₆ are both —H; R₁ andR₂ are both 3-fluorophenyl; R₃ and R₄ are both methyl; R₅ and R₆ areboth —H; R₁ and R₂ are both 4-chlorophenyl; R₃ and R₄ are both methyl;R₅ is methyl; R₆ is —H; R₁ and R₂ are both 2-dimethoxyphenyl; R₃ and R₄are both methyl; R₅ and R₆ are both —H; R₁ and R₂ are both3-methoxyphenyl; R₃ and R₄ are both methyl; R₅ and R₆ are both —H; R₁and R₂ are both 2,3-dimethoxyphenyl; R₃ and R₄ are both methyl; R₅ andR₆ are both —H; R₁ and R₂ are both 2,3-dimethoxyphenyl; R₃ and R₄ areboth methyl; R₅ is methyl; R₆ is —H; R₁ and R₂ are both2,5-difluorophenyl; R₃ and R₄ are both methyl; R₅ and R₆ are both —H; R₁and R₂ are both 2,5-difluorophenyl; R₃ and R₄ are both methyl; R₅ ismethyl; R₆ is —H; R₁ and R₂ are both 2,5-dichlorophenyl; R₃ and R₄ areboth methyl; R₅ and R₆ are both —H; R₁ and R₂ are both2,5-dimethylphenyl; R₃ and R₄ are both methyl; R₅ and R₆ are both —H; R₁and R₂ are both 2,5-dimethoxyphenyl; R₃ and R₄ are both methyl; R₅ andR₆ are both —H; R₁ and R₂ are both phenyl; R₃ and R₄ are both methyl; R₅and R₆ are both —H; R₁ and R₂ are both 2,5-dimethoxyphenyl; R₃ and R₄are both methyl; R₅ is methyl; R₆ is —H; R₁ and R₂ are both cyclopropyl;R₃ and R₄ are both methyl; R₅ and R₆ are both —H; R₁ and R₂ are bothcyclopropyl; R₃ and R₄ are both ethyl; R₅ and R₆ are both —H; R₁ and R₂are both cyclopropyl; R₃ and R₄ are both methyl; R₅ is methyl; R₆ is —H;R₁ and R₂ are both 1-methylcyclopropyl; R₃ and R₄ are both methyl; R₅and R₆ are both —H; R₁ and R₂ are both 1-methylcyclopropyl; R₃ and R₄are both methyl; R₅ is methyl and R₆ is —H; R₁ and R₂ are both1-methylcyclopropyl; R₃ and R₄ are both methyl; R₅ is ethyl and R₆ is—H; R₁ and R₂ are both 1-methylcyclopropyl; R₃ and R₄ are both methyl;R₅ is n-propyl and R₆ is —H; R₁ and R₂ are both 1-methylcyclopropyl; R₃and R₄ are both methyl; R₅ and R₆ are both methyl; R₁ and R₂ are both1-methylcyclopropyl; R₃ and R₄ are both ethyl; R₅ and R₆ are both —H; R₁and R₂ are both 1-methylcyclopropyl; R₃ is methyl, and R₄ is ethyl; R₅and R₆ are both —H; R₁ and R₂ are both 2-methylcyclopropyl; R₃ and R₄are both methyl; R₅ and R₆ are both —H; R₁ and R₂ are both2-phenylcyclopropyl; R₃ and R₄are both methyl; R₅ and R₆ are both —H; R₁and R₂ are both 1-phenylcyclopropyl; R₃ and R₄ are both methyl; R₅ andR₆ are both —H; R₁ and R₂ are both cyclobutyl; R₃ and R₄ are bothmethyl; R₅ and R₆ are both —H; R₁ and R₂ are both cyclopentyl; R₃ and R₄are both methyl; R₅ and R₆ are both —H; R₁ and R₂ are both cyclohexyl;R₃ and R₄ are both methyl; R₅ and R₆ are both —H; R₁ and R₂ are bothcyclohexyl; R₃ and R₄are both phenyl; R₅ and R₆ are both —H; R₁ and R₂are both methyl; R₃ and R₄ are both methyl; R₅ and R₆ are both —H; R₁and R₂ are both methyl; R₃ and R₄ are both t-butyl; R₅ and R₆ are both—H; R₁ and R₂ are both methyl; R₃ and R₄ are both phenyl; R₅ and R₆ areboth —H; R₁ and R₂ are both t-butyl; R₃ and R₄are both methyl; R₅ and R₆are both —H; R₁ and R₂ are ethyl; R₃ and R₄ are both methyl; R₅ and R₆are both —H; or R₁ and R₂ are both n-propyl; R₃ and R₄ are both methyl;R₅ and R₆ are both —H.
 30. A method of preparing a bis(thio-hydrazideamide) disalt, comprising the steps of: combining a neutralbis(thio-hydrazide amide), an organic solvent and a base to form abis(thio-hydrazide amide) solution; and combining the solution and anorganic antisolvent, thereby precipitating a disalt of thebis(thio-hydrazide amide) from the bis(thio-hydrazide amide) solution.31. The method of claim 30, wherein the neutral bis(thio-hydrazideamide) is represented by the following Structural Formula:

wherein: Y is a covalent bond or an optionally substituted straightchained hydrocarbyl group; R₁-R₄ are independently —H, an optionallysubstituted aliphatic group, an optionally substituted aryl group, or R₁and R₃ taken together with the carbon and nitrogen atoms to which theyare bonded, and/or R₂ and R₄ taken together with the carbon and nitrogenatoms to which they are bonded, form a non-aromatic heterocyclic ringoptionally fused to an aromatic ring; and Z is O or S.
 32. The method ofclaim 24, wherein the subject is a human.
 33. A method of treating asubject with melanoma, said method comprising administering to thesubject an effective amount of a compound represented by the followingstructural formula:

in combination with paclitaxel or an analog of paclitaxel.
 34. Themethod of claim 33, wherein the subject is a human.